Anthracycline derivative conjugates, process for their preparation and their use as antitumor compounds

ABSTRACT

The present invention relates to conjugates of therapeutically useful anthracyclines with carriers such as polyclonal and monoclonal antibodies, proteins or peptides of natural or synthetic origin; methods for their preparation, pharmaceutical composition containing them and use thereof in treating certain mammalian tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/502,433 filed on14 Jul. 2009, and also claims the benefit under 35 USC §119(e) of U.S.Provisional Application Ser. No. 61/080,944 filed on 15 Jul. 2008, whichis incorporated by reference in entirety.

FIELD OF THE INVENTION

The present invention relates to conjugates of therapeutically usefulanthracyclines with carriers such as polyclonal and monoclonalantibodies, proteins or peptides of natural or synthetic origin; methodsfor their preparation, pharmaceutical composition containing them anduse thereof in treating certain mammalian tumors. The invention alsorelates to new anthracycline derivatives and to their preparation.

BACKGROUND OF THE INVENTION

Anthracyclines are antibiotic compounds that exhibit cytotoxic activity.Studies have indicated that anthracyclines may operate to kill cells bya number of different mechanisms including: 1) intercalation of the drugmolecules into the DNA of the cell thereby inhibiting DNA-dependentnucleic acid synthesis; 2) production by the drug of free radicals whichthen react with cellular macromolecules to cause damage to the cells or3) interactions of the drug molecules with the cell membrane [see, e.g.,C. Peterson et al., “Transport And Storage Of Anthracycline InExperimental Systems And Human Leukemia” in Anthracycline Antibiotics InCancer Therapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102].Because of their cytotoxic potential anthracyclines have been used inthe treatment of numerous cancers such as leukemia, breast carcinoma,lung carcinoma, ovarian adenocarcinoma and sarcomas [see e.g., P.H-Wiernik, in Anthracycline: Current Status And New Developments p 11].Commonly used anthracyclines include doxorubicin, epirubicin, idarubicinand daunomycin.

In the recent years many new highly cytotoxic anthracyclines have beensynthesized. For example nemorubicin, the anthracycline derivativebearing a substituted morpholino ring linked to the C-3′ position of thesugar moiety has shown promising antitumor activity on experimentalmurine tumors [see: J. W. Lown, Bioactive Molecules (1988) vol 6:55-101]and is currently under clinical phase trials for the treatment ofhepatocellular carcinoma [see: C. Sessa, O. Valota, C. Geroni,Cardiovascular Toxicology (2007) 7(2):75-79]. Although these compoundsmay be useful in the treatment of neoplasm and other disease stateswherein a selected cell population is sought to be eliminated, theirtherapeutic efficacy is often limited by the dose-dependent toxicityassociated with their administration.

Attempts to improve the therapeutic effect of these compounds have beentried by linking the anthracycline to antibodies or to differentcarriers. An example of an anthracycline conjugated with antibodies isreported, for example, in EP 0328147 to Bristol Myers, in WO 9202255 toFarmitalia Carlo Erba or in U.S. Pat. No. 5,776,458 to Pharmacia &Upjohn.

Other interesting tricyclic morpholino anthracycline derivatives,characterized by high activity, were described and claimed in theInternational patent application WO 98/02446 (1997) of M. Caruso et al.Among these derivatives, a particularly active compound is PNU-159682,described by Quintieri, L., Geroni, C. et al. in Clinical CancerResearch (2005) 11(4):1608-1617. Compound PNU-159682 has the formula(IIA) as defined herein below, and the following chemical names:

-   5,12-naphthacenedione,    7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-[[(1S,3R,4aS,9S,9aR,10aS)-octahydro-9-methoxy-1-methyl-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy]-,    (8S,10S)-(9CI);-   5,12-naphthacenedione,    7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-[(octahydro-9-methoxy-1-methyl-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy]-,    [1S-[1α,3β(8R*,10R*),4aβ,9α,9aα,10aβ]] or    (8S,10S)-6,8,1′-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-7,8,9,10-tetrahydrotetracene-5,12-dione.

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders (Carter, P.(2006) Nature Reviews Immunology 6:343-357). The use of antibody-drugconjugates (ADC), i.e. immunoconjugates, for the local delivery ofcytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumorcells in the treatment of cancer, targets delivery of the drug moiety totumors, and intracellular accumulation therein, whereas systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Xie et al (2006) Expert. Opin. Biol.Ther. 6(3):281-291; Kovtun et al (2006) Cancer Res. 66(6):3214-3121; Lawet al (2006) Cancer Res. 66(4):2328-2337; Wu et al (2005) NatureBiotech. 23(9):1137-1145; Lambert J. (2005) Current Opin. in Pharmacol.5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103;Payne, G. (2003) Cancer Cell 3:207-212; Trail et al (2003) CancerImmunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) AnticancerResearch 19:605-614). Maximal efficacy with minimal toxicity is soughtthereby. Efforts to design and refine ADC have focused on theselectivity of monoclonal antibodies (mAbs) as well as drug mechanism ofaction, drug-linking, drug/antibody ratio (loading), and drug-releasingproperties (McDonagh (2006) Protein Eng. Design & Sel.; Doronina et al(2006) Bioconj. Chem. 17:114-124; Erickson et al (2006) Cancer Res.66(8):1-8; Sanderson et al (2005) Clin. Cancer Res. 11:843-852; Jeffreyet al (2005) J. Med. Chem. 48:1344-1358; Hamblett et al (2004) Clin.Cancer Res. 10:7063-7070). Drug moieties may impart their cytotoxic andcytostatic effects by mechanisms including tubulin binding, DNA binding,or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive orless active when conjugated to large antibodies or protein receptorligands.

The anthracycline analog, doxorubicin (ADRIAMYCIN®) is thought tointeract with DNA by intercalation and inhibition of the progression ofthe enzyme topoisomerase II, which unwinds DNA for transcription.Doxorubicin stabilizes the topoisomerase II complex after it has brokenthe DNA chain for replication, preventing the DNA double helix frombeing resealed and thereby stopping the process of replication.Doxorubicin and daunorubicin (DAUNOMYCIN) are prototype cytotoxicnatural product anthracycline chemotherapeutics (Sessa et al (2007)Cardiovasc. Toxicol. 7:75-79). Immunoconjugates and prodrugs ofdaunorubicin and doxorubicin have been prepared and studied (Kratz et al(2006) Current Med. Chem. 13:477-523; Jeffrey et al (2006) Bioorganic &Med. Chem. Letters 16:358-362; Torgov et al (2005) Bioconj. Chem.16:717-721; Nagy et al (2000) Proc. Natl. Acad. Sci. 97:829-834;Dubowchik et al (2002) Bioorg. & Med. Chem. Letters 12:1529-1532; Kinget al (2002) J. Med. Chem. 45:4336-4343; U.S. Pat. No. 6,630,579). Theantibody-drug conjugate BR96-doxorubicin reacts specifically with thetumor-associated antigen Lewis-Y and has been evaluated in phase I andII studies (Saleh et al (2000) J. Clin. Oncology 18:2282-2292; Ajani etal (2000) Cancer Jour. 6:78-81; Tolcher et al (1999) J. Clin. Oncology17:478-484).

Morpholino analogs of doxorubicin and daunorubicin, formed bycyclization on the glycoside amino group, have greater potency (Acton etal (1984) J. Med. Chem. 638-645; U.S. Pat. No. 4,464,529; U.S. Pat. No.4,672,057; U.S. Pat. No. 5,304,687). Nemorubicin is a semisyntheticanalog of doxorubicin with a 2-methoxymorpholino group on the glycosideamino of doxorubicin and has been under clinical evaluation (Grandi etal (1990) Cancer Treat. Rew. 17:133; Ripamonti et al (1992) Brit. J.Cancer 65:703), including phase II/III trials for hepatocellularcarcinoma (Sun et al (2003) Proceedings of the American Society forClinical Oncology 22, Abs1448; Quintieri (2003) Proceedings of theAmerican Association of Cancer Research, 44:1st Ed, Abs 4649; Pacciariniet al (2006) Jour. Clin. Oncology 24:14116)

Nemorubicin is named as(8S,10S)-6,8,11-trihydroxy-10-((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholino)-6-methyltetrahydro-2H-pyran-2-yloxy)-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione,with CAS Reg. No. 108852-90-0, and has the structure:

Several metabolites of nemorubicin (MMDX) from liver microsomes havebeen characterized, including PNU-159682, (Quintieri et al (2005)Clinical Cancer Research, 11(4):1608-1617; Beulz-Riche et al (2001)Fundamental & Clinical Pharmacology, 15(6):373-378; EP 0889898; WO2004/082689; WO 2004/082579). PNU-159682 was remarkably more cytotoxicthan nemorubicin and doxorubicin in vitro, and was effective in vivotumor models. PNU-159682 (formula (IIA) is named as3′-deamino-3″,4′-anhydro-[2″(S)-methoxy-3″(R)-oxy-4″-morpholinyl]doxorubicin,and has the structure:

Certain PNU-159682 antibody-drug conjugates have been described(“NEMORUBICIN METABOLITE AND ANALOG ANTIBODY-DRUG CONJUGATES ANDMETHODS”, PCT/US2009/031199, filed 16 Jan. 2009).

SUMMARY OF THE INVENTION

An aspect of the present invention is to provide new anthracyclinederivative conjugates with carriers such as monoclonal or polyclonalantibodies reactive with a selected cell population, proteins, peptidesor other carriers of synthetic origin reactive with receptor tissues.

Another aspect is a process for the preparation of such conjugates aswell as useful intermediates.

The conjugates of the present invention are characterized by the formula(I)

[Ant-L-Z-]_(m)-T  (I)

wherein

Ant is anthracycline derivative residue,

L is a linker,

Z is a spacer,

m is an integer of from 1 to 30 and

T is carrier such as a protein, peptide, monoclonal or polyclonalantibody or a chemically modified derivative thereof suitable to beattached to the [Ant-L-Z-] moiety or moieties, or a polymeric carrier;

characterized in that the anthracycline derivative residue that Antrepresents can be released to give an anthracycline derivative offormula (II):

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof.

The anthracycline derivative residue is tethered to the carrier througha linker spacer [L-Z-], and the bond between the anthracyclinederivative and the linker arm can be cleaved under physiologicalconditions so that to release an anthracycline derivative of formula(II) as defined above, that is the bioactive agent.

For example, conjugates wherein the bond between the anthracyclinederivative and the linker is sensitive to acid conditions or to reducingconditions can release the anthracycline derivative in the conditionstypically encountered within the cell, e.g., in lysosomal vesicles.

A preferred method of the present invention is to treat specific typesof cancer including but not limited to: carcinoma such as bladder,breast, colon, kidney, liver, lung, including small cell lung cancer,esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid,prostate, and skin, including squamous cell carcinoma; hematopoietictumors of lymphoid lineage including leukaemia, acute lymphociticleukaemia, acute lymphoblastic leukaemia, B-cell lymphoma,T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy celllymphoma and Burkett's lymphoma; hematopoietic tumors of myeloidlineage, including acute and chronic myelogenous leukemias,myelodysplastic syndrome and promyelocytic leukaemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumorsof the central and peripheral nervous system, including astrocytomaneuroblastoma, glioma and schwannomas; other tumors, including melanoma,seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum,keratoxanthoma, thyroid follicular cancer and Kaposi's sarcoma.

Another preferred method of the present invention is to treat specificcellular proliferation disorders such as, for example, benign prostatehyperplasia, familial adenomatosis polyposis, neurofibromatosis,psoriasis, vascular smooth cell proliferation associated withatherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis andpost-surgical stenosis and restenosis.

The anthracycline derivative conjugates of the formula (I) can beprepared through a process consisting of standard synthetictransformations; such process and the intermediates used in such processare also provided by the present invention.

The present invention also provides a pharmaceutical compositioncomprising an anthracycline derivative conjugate of the formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient or diluent.

An aspect of the invention is an anthracycline derivative of formula(IIc)

Ant-L-(Z)_(m)—X  (IIc)

wherein Ant is an anthracycline derivative selected from the structures:

where the wavy line indicates the attachment to linker L; Z is anoptional spacer; m is 0 or 1; X is a reactive functional group; and n isan integer from 1 to 6.

An aspect of the invention is an antibody-drug conjugate (ADC) compoundcomprising an antibody covalently attached by a linker L and an optionalspacer Z to one or more anthracycline derivative drug moieties D, thecompound having formula (Ic)

Ab-(L-Z_(m)-D)_(p)  (Ic)

or a pharmaceutically acceptable salt thereof, wherein p is an integerfrom 1 to 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in a graph form the stability of the compound 2 shown inTable 1 at pH 5.2 wherein in (Y) axis there is the percentage amount ofthe compound of the formula (IIA) as defined below, and in (X) axis thetime in hours. This graph demonstrates the acid-sensitivity of theacetalic bound of the invention as indicated by the increased release ofthe free compound of the formula (IIA) from the conjugate under acid pH.

FIG. 2 shows an exemplary process to prepare Formula Ia compounds byreacting anthracycline derivative Formula II with vinyl ether compounds(X) or (IX) to give acetal compound (XI), hydrolysis to a carboxycompound (XII), and activation to form an N-hydroxy succinimide (NHS)ester (XIII), ready for conjugation with a carrier compound.

FIG. 3 a shows an exemplary process to prepare Formula Ia compounds byreaction of activated N-hydroxy succinimide (NHS) ester (XIII) withamino-thiol reagent (XXI) to give XXII which can be reacted withpyridyl-disulfide carrier (T) intermediate (VI) to give disulfide linkedanthracycline derivative conjugate (Ia), or reacted with maleimidecarrier (T) intermediate (XXII) to give maleimide linked anthracyclinederivative conjugate (Ia).

FIG. 3 b shows exemplary processes to prepare Formula Ia compounds byreaction of activated N-hydroxy succinimide (NHS) ester (XIII) withamino-ester reagent (XVIII), followed by ester hydrolysis to givecarboxy anthracycline derivative (XIX), which can be activated as an NHSester and coupled with amino carrier T intermediate, e.g. an antibody,to give amide linked anthracycline derivative conjugate (Ia).

FIG. 3 c shows exemplary processeses to prepare Formula Ia compounds byreaction of activated N-hydroxy succinimide (NHS) ester (XIII) withamino or thiol group of carrier T (XIV), e.g. antibody to give amide orthioamide linked anthracycline derivative conjugate (Ia)

FIG. 4 shows an exemplary process to react an anthracycline derivative(II) with an acyl hydrazide derivative (XXV) to form hydrazone (XXVI)followed by conjugation with a carrier T reagent to give anthracyclinederivative conjugate (Ib).

FIG. 5 a shows exemplary processes: (2a) reacting an anthracyclinederivative hydrazone (XXVI) with a thiol- or amino-carrier T compound(XIV) to give an anthracycline derivative conjugate (Ib); (2b) reactingan anthracycline derivative hydrazone (XXVI) with reagent (XXVII),followed by deprotection to (XXVIII) and coupling with carboxyl-carrierT compound (XVII) to give an anthracycline derivative conjugate (Ib);and (2d) condensation of deprotected (XXVIII) with aldehyde-carrier Tcompound (XX) to give anthracycline derivative conjugate (Ib).

FIG. 5 b shows an exemplary process: (2c) reacting an anthracyclinederivative hydrazone (XXVI) with reagent (XXIX), followed bydeprotection and coupling with amino-carrier T compound to giveanthracycline derivative conjugate (Ib)

FIG. 5 c shows exemplary processes: (2e) reacting an anthracyclinederivative hydrazone (XXVI) with reagent (XXXI) to give (XOCH), followedby (2e″) coupling with pyridyl disulfide carrier compound (VI) to giveanthracycline derivative conjugate (Ib), and (2e′) coupling (XXXII) withmaleimide carrier compound (V) to give anthracycline derivativeconjugate (Ib).

FIG. 6 shows an exemplary process to react an anthracycline derivative(II) with a acyl hydrazide, pyridyl disulfide (XXVIII) to form pyridyldisulfide hydrazone (XXXIV) followed by conjugation with a carrier Treagent to give anthracycline derivative conjugate (Ib).

FIG. 7 a shows exemplary processeses: (3a) reacting an anthracyclinederivative (XXXIV) with a thiol-carrier (XIV) to give anthracyclinederivative conjugate (Ib); (3b) reacting an anthracycline derivative(XXXIV) with a thiol compound (XXXV) to give amine disulfide compound(XXXVI) which is coupled with a carboxyl-carrier T reagent to giveanthracycline derivative conjugate (Ib); and (3d) condensation ofdeprotected (XXXVI) with aldehyde-carrier T compound (XX) to giveanthracycline derivative conjugate (Ib).

FIG. 7 b shows an exemplary process (3c) reacting an anthracyclinederivative (XXXIV) with thiol ester (XXXVII), followed deprotection tocarboxyl disulfide compound (XXXVIII), and coupling with amino-carrier T(XIV) to give anthracycline derivative conjugate (Ib).

FIG. 7 c shows exemplary processes: (3e) reacting an anthracyclinederivative (XXXIV) thiol reagent (XXXIX) to give disulfide thiol (XL),(3e′) coupling disulfide thiol (XL) with pyridyl disulfide carriercompound (VI) to give anthracycline derivative conjugate (Ib); andcoupling disulfide thiol (XL) with maleimide carrier compound (V) togive anthracycline derivative conjugate (Ib).

FIG. 7 d shows a synthetic route to drug-linker intermediateN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-{[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]carbonyl}piperazin-1-yl)carbonyl]oxy}methyl)phenyl]-L-ornithinamide55

FIG. 8 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusnM concentrations of: free drug PNU-159682 continuous exposure,PNU-159682 1 hr incubation, NHS-ketal-Ant 50, maleimide-ketal-Ant 51,maleimide-hydrazone-Ant 52, and thiopyridine-hydrazone-Ant 53.

FIG. 9 shows a plot of BT-474 in vitro cell viability at 3 days versusnM concentrations of: free drug PNU-159682 continuous exposure,PNU-159682 1 hr incubation, NHS-ketal-Ant 50, maleimide-ketal-Ant 51,maleimide-hydrazone-Ant 52, and thiopyridine-hydrazone-Ant 53.

FIG. 10 shows a plot of BT-474 in vitro cell viability at 3 days versusnM concentrations of: free drug PNU-159682 continuous exposure,PNU-159682 1 hr incubation, NHS-ketal-Ant 50, maleimide-ketal-Ant 51,maleimide-hydrazone-Ant 52, and thiopyridine-hydrazone-Ant 53.

FIG. 11 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus nM concentrations of: free drugPNU-159682 continuous exposure, PNU-159682 1 hr incubation,NHS-ketal-Ant 50, maleimide-ketal-Ant 51, maleimide-hydrazone-Ant 52,and thiopyridine-hydrazone-Ant 53. The DoxRes Her2 cell line is alsoknown as “AdrRes Her2”.

FIG. 12 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102, thio-trastuzumab(HC A114C)-maleimide hydrazone-Ant 103. (Heavy chain antibody number byKabat numbering scheme)

FIG. 13 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102, thio-trastuzumab(HC A114C)-maleimide hydrazone-Ant 103.

FIG. 14 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102, thio-trastuzumab(HC A114C)-maleimide hydrazone-Ant 103.

FIG. 15 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus concentrations of: trastuzumab,trastuzumab-MCC-DM1 101, thio-trastuzumab (HC A114C)-maleimide ketal-Ant102, thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103.

FIG. 16 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: anti-CD22 NHS ketal-Ant 110, trastuzumab,trastuzumab-MCC-DM1 101, thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105.

FIG. 17 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: anti-CD22 NHS ketal-Ant 110, trastuzumab,trastuzumab-MCC-DM1 101, thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105.

FIG. 18 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: anti-CD22 NHS ketal-Ant 110, trastuzumab,trastuzumab-MCC-DM1 101, thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105.

FIG. 19 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus concentrations of: anti-CD22 NHSketal-Ant 110, trastuzumab, trastuzumab-MCC-DM1 101, thio-trastuzumab(HC A114C)-NHS-ketal-Ant 105.

FIG. 20 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations (μg/ml) of: thio-trastuzumab (HC A114C)-maleimideketal-Ant 102, thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103,thio-trastuzumab (HC A114C)-thiopyridine hydrazone-Ant 104,thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-MC-vc-PAB-MMAE 106.

FIG. 21 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, thio-anti-CD22 (HC A114C)-maleimideketal-Ant 107, thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108,thio-anti-CD22 (HC A114C)-thiopyridine hydrazone-Ant 109,anti-CD22-NHS-ketal-Ant 110, PNU-159682 free drug.

FIG. 22 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102,thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103,thio-trastuzumab (HC A114C)-thiopyridine hydrazone-Ant 104,thio-trastuzumab (HC A114C)—NHS-ketal-Ant 105, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-MC-vc-PAB-MMAE 106.

FIG. 23 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, thio-anti-CD22 (HC A114C)-maleimideketal-Ant 107, thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108,thio-anti-CD22 (HC A114C)-thiopyridine hydrazone-Ant 109,anti-CD22-NHS-ketal-Ant 110, PNU-159682 free drug.

FIG. 24 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102,thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103,thio-trastuzumab (HC A114C)-thiopyridine hydrazone-Ant 104,thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-MC-vc-PAB-MMAE 106.

FIG. 25 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, thio-anti-CD22 (HC A114C)-maleimideketal-Ant 107, thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108,thio-anti-CD22 (HC A114C)-thiopyridine hydrazone-Ant 109,anti-CD22-NHS-ketal-Ant 110, PNU-159682 free drug.

FIG. 26 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus concentrations of: thio-trastuzumab (HCA114C)-maleimide ketal-Ant 102, thio-trastuzumab (HC A114C)-maleimidehydrazone-Ant 103, thio-trastuzumab (HC A114C)-thiopyridinehydrazone-Ant 104, thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105,trastuzumab-MCC-DM1 101, thio-trastuzumab (HC A114C)-MC-vc-PAB-MMAE 106.

FIG. 27 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus concentrations of: trastuzumab,thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107, thio-anti-CD22 (HCA114C)-maleimide hydrazone-Ant 108, thio-anti-CD22 (HCA114C)-thiopyridine hydrazone-Ant 109, anti-CD22-NHS-ketal-Ant 110,PNU-159682 free drug.

FIG. 28 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus concentrations of: thio-trastuzumab (HCA114C)-maleimide ketal-Ant 102, thio-trastuzumab (HC A114C)-maleimidehydrazone-Ant 103, thio-trastuzumab (HC A114C)-thiopyridinehydrazone-Ant 104, thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105,trastuzumab-MCC-DM1 101, thio-trastuzumab (HC A114C)-MC-vc-PAB-MMAE 106.

FIG. 29 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus concentrations of: trastuzumab,thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107, thio-anti-CD22 (HCA114C)-maleimide hydrazone-Ant 108, thio-anti-CD22 (HCA114C)-thiopyridine hydrazone-Ant 109, anti-CD22-NHS-ketal-Ant 110,PNU-159682 free drug, all plus verapamil.

FIG. 30 shows a plot of the in vivo mean tumor volume change over timein Burkitt's lymphoma Bjab-luc xenograft tumors inoculated into CB17SCID mice after single intravenous (iv) dosing on day 0 with: (1)Vehicle, (2) thio-anti-CD22 (HC A114C)-MC-vc-PAB-MMAE 111 1 mg/kg, (3)thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102 1 mg/kg, (4)thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102 5 mg/kg, (5)thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107 1 mg/kg, (6)thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107 5 mg/kg, (7)thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103 1 mg/kg, (8)thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108 1 mg/kg, (9)PNU-159682 free drug 8.77 ug/kg (26 ug/m2 exposure), matched to the drugdose of 1 mg/kg antibody-drug conjugates.

FIG. 31 shows a plot of the in vivo mean tumor volume change over timein MMTV-HER2Fo5 mammary allograft tumors inoculated into CRL nu/nu miceafter single iv dosing on day 0 with: (1) Vehicle, (2)trastuzumab-MCC-DM1 101 5/mg/kg, (3) trastuzumab-MCC-DM1 101 10 mg/kg,(4) thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102 5 mg/kg, (5)thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102 10 mg/kg, (6)thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107 5 mg/kg, (7)thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107 10 mg/kg, (8)trastuzumab-MCC-DM1 101 5 mg/kg+thio-trastuzumab (HC A114C)-maleimideketal-Ant 102 5 mg/kg.

FIG. 32 shows a plot of the in vivo mean tumor volume change over timein LnCap-Ner xenograft tumors inoculated into male SCID-beige mice aftersingle iv dosing on day 0 with: (1) Vehicle, (2) thio-anti-steap1 (HCA114C)-MC-vc-PAB-MMAE 112 1 mg/kg, (3) thio-anti-steap1 (HCA114C)-MC-vc-PAB-MMAE 112 3 mg/kg, (4) thio-anti-steap1 (HCA114C)-maleimide ketal-Ant 113 1 mg/kg, (5) thio-anti-steap1 (HCA114C)-maleimide ketal-Ant 113 3 mg/kg, (6) thio-anti-steap1 (HCA114C)-maleimide ketal-Ant 113 6 mg/kg, (7) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 1 mg/kg, (8) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 3 mg/kg, (9) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 6 mg/kg

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims. One skilled in theart will recognize many methods and materials similar or equivalent tothose described herein, which could be used in the practice of thepresent invention. The present invention is in no way limited to themethods and materials described. Unless defined otherwise, technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs, and are consistent with: Singleton et al., (1994) Dictionary ofMicrobiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York,N.Y.; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York.

DEFINITIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings.

When trade names are used herein, applicants intend to independentlyinclude the trade name product formulation, the generic drug, and theactive pharmaceutical ingredient(s) of the trade name product.

“Anthracycline derivative” is a nemorubicin metabolite, or analogcompound, including but not limited to PNU-159682.

“Anthracycline derivative conjugate” is a compound comprised of ananthracycline derivative covalently attached through a linker to acarrier moiety, including antibodies, proteins or peptides.Anthracycline derivative conjugate compounds include antibody-drugconjugate (ADC) compounds.

The term “amino acid side chain” includes those groups found in: (i)naturally occurring amino acids such as alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine; (ii) minor amino acids suchas ornithine and citrulline; (iii) unnatural amino acids, beta-aminoacids, synthetic analogs and derivatives of naturally occurring aminoacids; and (iv) all enantiomers, diastereomers, isomerically enriched,isotopically labelled (e.g. ²H, 3H, ¹⁴C, ¹⁵N), protected forms, andracemic mixtures thereof.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin can be of anytype (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Theimmunoglobulins can be derived from any species, including human,murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodiesinclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g., OldWorld Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

An “ErbB receptor” is a receptor protein tyrosine kinase which belongsto the ErbB receptor family which are important mediators of cellgrowth, differentiation and survival. The ErbB receptor family includesfour distinct members including epidermal growth factor receptor (EGFR,ErbB1, HER1), HER2 (ErbB2 or p185^(neu)), HER3 (ErbB3) and HER4 (ErbB4or tyro2). The ErbB receptor will generally comprise an extracellulardomain, which may bind an ErbB ligand; a lipophilic transmembranedomain; a conserved intracellular tyrosine kinase domain; and acarboxyl-terminal signaling domain harboring several tyrosine residueswhich can be phosphorylated. The ErbB receptor may be a “nativesequence” ErbB receptor or an “amino acid sequence variant” thereof. TheErbB receptor may be native sequence human ErbB receptor. Accordingly, a“member of the ErbB receptor family” is EGFR (ErbB1), ErbB2, ErbB3,ErbB4 or any other ErbB receptor currently known or to be identified inthe future. Sequence identity screening has resulted in theidentification of two other ErbB receptor family members; ErbB3 (U.S.Pat. No. 5,183,884; U.S. Pat. No. 5,480,968; Kraus et al (1989) PNAS(USA) 86:9193-9197) and ErbB4 (EP 599274; Plowman et al (1993) Proc.Natl. Acad. Sci. USA, 90:1746-1750; and Plowman et al (1993) Nature366:473-475). Both of these receptors display increased expression on atleast some breast cancer cell lines. Anti-ErbB2 antibodies have beencharacterized (U.S. Pat. No. 5,677,171; U.S. Pat. No. 5,821,337; U.S.Pat. No. 6,054,297; U.S. Pat. No. 6,165,464; U.S. Pat. No. 6,407,213;U.S. Pat. No. 6,719,971; U.S. Pat. No. 6,800,738; Fendly et al (1990)Cancer Research 50:1550-1558; Kotts et al. (1990) In Vitro 26(3):59A;Sarup et al. (1991) Growth Regulation 1:72-82; Shepard et al. J. (1991)Clin. Immunol. 11(3):117-127; Kumar et al. (1991) Mol. Cell. Biol.11(2):979-986; Lewis et al. (1993) Cancer Immunol. Immunother.37:255-263; Pietras et al. (1994) Oncogene 9:1829-1838; Vitetta et al.(1994) Cancer Research 54:5301-5309; Sliwkowski et al. (1994) J. Biol.Chem. 269(20):14661-14665; Scott et al. (1991) J. Biol. Chem.266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994) 91:7202-7206;Lewis et al. (1996) Cancer Research 56:1457-1465; and Schaefer et al.(1997) Oncogene 15:1385-1394.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all, or substantially all, ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al (1986) Nature, 321:522-525; Riechmann et al(1988) Nature 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol.,2:593-596). Humanized anti-ErbB2 antibodies include huMAb4D5-1,huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7and huMAb4D5-8 (HERCEPTIN®, trastuzumab) as described in Table 3 of U.S.Pat. No. 5,821,337 expressly incorporated herein by reference; humanized520C9 (WO 93/21319) and humanized 2C4 antibodies.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

A “disorder” is any condition that would benefit from treatment of thepresent invention. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include benign and malignant tumors; leukemia andlymphoid malignancies, in particular breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic,prostate or bladder cancer; neuronal, glial, astrocytal, hypothalamicand other glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders. Anexemplary disorder to be treated in accordance with the presentinvention is a solid, malignant tumor.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may: (i)reduce the number of cancer cells; (ii) reduce the tumor size; (iii)inhibit, retard, slow to some extent and preferably stop cancer cellinfiltration into peripheral organs; (iv) inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; (v) inhibit tumor growth;and/or (vi) relieve to some extent one or more of the symptomsassociated with the cancer. To the extent the drug may prevent growthand/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. In animal models, efficacy may be assessed by physicalmeasurements of the tumor during the course following administration ofthe ADC, and by determining partial and complete remission of tumor. Forcancer therapy, efficacy can, for example, be measured by assessing thetime to disease progression (TTP) and/or determining the response rate(RR).

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, gastrointestinal stromal tumor (GIST),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, aswell as head and neck cancer.

An “ErbB-expressing cancer” is one comprising cells which have ErbBprotein present at their cell surface. An “ErbB2-expressing cancer” isone which produces sufficient levels of ErbB2 at the surface of cellsthereof, such that an anti-ErbB2 antibody can bind thereto and have atherapeutic effect with respect to the cancer.

A cancer which “overexpresses” a receptor, e.g. an ErbB receptor, is onewhich has significantly higher levels of the receptor, such as ErbB2, atthe cell surface thereof, compared to a noncancerous cell of the sametissue type. Such overexpression may be caused by gene amplification orby increased transcription or translation. Receptor overexpression maybe determined in a diagnostic or prognostic assay by evaluatingincreased levels of the receptor protein present on the surface of acell (e.g., via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of receptor-encoding nucleic acidin the cell, e.g., via fluorescent in situ hybridization (FISH; see WO98/45479), southern blotting, or polymerase chain reaction (PCR)techniques, such as real time quantitative PCR (RT-PCR). Overexpressionof the receptor ligand, may be determined diagnostically by evaluatinglevels of the ligand (or nucleic acid encoding it) in the patient, e.g.,in a tumor biopsy or by various diagnostic assays such as the IHC, FISH,southern blotting, PCR or in vivo assays described above. One may alsostudy receptor overexpression by measuring a shed antigen (e.g., ErbBextracellular domain) in a biological fluid such as serum (see, e.g.,U.S. Pat. No. 4,933,294; WO 91/05264; U.S. Pat. No. 5,401,638; and Siaset al (1990) J. Immunol. Methods 132: 73-80). Aside from the aboveassays, various other in vivo assays are available to the skilledpractitioner. For example, one may expose cells within the body of thepatient to an antibody which is optionally labeled with a detectablelabel, e.g., a radioactive isotope, and binding of the antibody to cellsin the patient can be evaluated, e.g., by external scanning forradioactivity or by analyzing a biopsy taken from a patient previouslyexposed to the antibody.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,²¹¹At, ¹³¹I, ¹²⁵I, ⁹Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, andradioactive isotopes of Lu), chemotherapeutic agents, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including synthetic analogs andderivatives thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkyatingagents, antimetabolites, spindle poison plant alkaloids,cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine,dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CASNo. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1 I, calicheamicin omegaI1 (AngewChem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the PI3K inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

“Alkyl” is C₁-C₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples of alkyl radicals include, but not limitedto: methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl,—CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu,n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (1-Bu, i-butyl,—CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂)_(,) 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkylene radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkylene radical is one to eight carbon atoms(C₁-C8), or one to six carbon atoms (C₁-C₆). Examples of alkylene groupsinclude, but are not limited to, methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted, and includesradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations. Examples include, but are not limited to,ethylenylene or vinylene (—CH═CH—), allyl (—CH₂CH═CH—), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp triple bond, whereinthe alkynyl radical may be optionally. Examples include, but are notlimited to, ethynylene (—C≡C—), propynylene (propargylene, —CH₂C≡C—),and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 20 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen, phosphorusand sulfur, the remaining ring atoms being C, where one or more ringatoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocyclesare described in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring carbon atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups areoptionally substituted independently with one or more substituentsdescribed herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

“Linker” or “link” means a divalent chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches an antibodyto a drug moiety. In various embodiments of formula I, a linker isspecified as L.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of an ADC.Exemplary salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” refers to an association of one ormore solvent molecules and an ADC. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine.

Anthracycline Derivatives and Anthracycline Derivative Conjugates

As stated before, in a first aspect the present invention relates toconjugates of an anthracycline derivative of the formula (I):

[Ant-L-Z-]_(n)-T  (I)

wherein Ant, L, Z, m and T are as above defined, or a pharmaceuticallyacceptable salt thereof.

In a second aspect, there are provided anthracycline derivatives of theformula (I′)

Ant-L-Z-Q (I′)

wherein Ant, L and Z are as above defined, and Q is a hydrogen atom, aC₁-C₆ alkyl, C₃-C₆ cycloalkyl, phenyl or benzyl group, or apharmaceutically salt thereof.

Preferably, the anthracycline derivative residue that Ant represents canbe released to give an anthracycline derivative of formula (IIA):

In another preferred aspect, the present invention provides ananthracycline derivative conjugate or a pharmaceutically salt thereof ofthe formula (Ia):

wherein:

R₁, R₂, Z, m and T are as defined above and L₁ is a linker of formula(III) or (IV):

wherein B is a C₁-C₆ alkylene moiety optionally hetero interrupted, andv, j, k and y are independently 0 or 1.

It is clear that in this instance, the anthracycline derivative residuewhich Ant represents is tether to the linker L₁ through an acetalic bondthat involves the primary alcohol at the C-14 of the anthracyclineskeleton.

As stated above, an anthracycline derivative conjugate or apharmaceutically salt thereof of the formula (I) releases the desiredfree anthracycline derivative, as shown below for the preferredconjugates of the formula (Ia)

wherein R₁, R₂, L₁, Z, m and T are as above defined.

Preferably, Z is a spacer group like

a) —NH—,

b) —S—,

c) aminoalkylene, thioalkylene, aminocycloalkylene or thiocycloalkylenebearing a further thiol or amino group or a carboxylic residue,

d) a peptidic residue able to tether the L₁ linker to the T carrier byforming new bonds as e.g.; amide bonds, disulfide bonds.

T is preferably selected from a polyclonal antibody, or fragmentthereof, comprising an antigen binding site, capable of binding to atumor associated antigen; a monoclonal antibody, or fragment thereofcomprising an antigen binding site, capable of binding to an antigenpreferentially or selectively expressed on tumor cell populations; apeptide or protein capable optionally of preferentially or selectivelybinding to a tumor cell; or a chemically modified derivative thereofsuitable to be attached to the [Ant-L₁-Z-] moiety or moieties, or apolymeric carrier.

Particularly preferred compounds of formula (Ia) are those wherein thespacer group which Z represents is:

i) —NH—, that is [—Z-]_(m)T is derived from a carrier of the formulaT-[NH₂]_(m) wherein m is above defined;

ii) —S—, that is [—Z-]_(m)T is derived from a carrier of the formulaT-[SH]_(m) wherein m is above defined;

iii)-NH-D-NH—CO— wherein -D- is a C₁-C₆ alkylene, C₃-C₆ cycloalkylene or-D-NH— is a peptide residue constituted from 1 to 4 amino acids havingat least one free amino group, that is [-Z-]_(m)T is derived from acarrier of formula T-[COOH]_(m) wherein m is above defined;

iv) —NH-D-CO—NH— wherein -D- is as defined above or -D-CO— is a peptideresidue constituted from 1 to 4 amino acids having at least one freecarboxylic group, that is [-Z-]_(m)T is derived from a carrier offormula T-[NH₂]_(m) wherein m is above defined;

v) —NH-D-N═CH— wherein -D- is as defined above and -D-N— is as definedabove for -D-NH, that is [-Z-]_(m)T is derived from a carrier of formulaT-[CHO]_(m) wherein m is above defined;

yl)-NH-D-S—CH— wherein -D- is as defined above or -D-S— is a peptideresidue constituted from 1 to 4 amino acids having at least one freethiol group, that is [-Z-]_(m)-T is derived from a carrier derivative offormula (V);

wherein m is above defined;

vii)-NH-D-S—S— wherein -D- and -D-S— are as defined above, that is[-Z-]_(m)-T is derived from a carrier derivative of formula (VI):

wherein m is above defined.

In a further preferred aspect, the present invention provides ananthracycline derivative conjugate or a pharmaceutically salt thereof ofthe formula (Ib):

wherein R₁ and R₂, Z and T are as defined above and L₂ is a linker offormula (VII) or (VIII):

wherein n is an integer from 1 to 9.

In this case, the release of the desired free anthracycline derivativecan be schematically illustrated as follows from the preferredconjugates of the formula (Ib):

Particularly preferred compounds of the formula (Ib) are those wherein:

a) L₂ is a linker of formula (VII) as defined above and Z is a spacergroup which is:

viii) as defined above under point i);

ix) as defined above under point ii);

x) as defined above under point iii);

xi) as defined above under point iv);

xii) as defined above under point v);

xiii) as defined above under point vi);

xiv) as defined above under point vii);

xv) —S-D-NH—CO— wherein -D-NH—CO— is as defined above under point iii);

xvi) —S-D-CO—NH— wherein -D-CO—NH is as defined above under point iv);

xvii) —S-D-N═C— wherein D and D-N— are as defined above under point v);

xviii) S-D-S—CH— wherein D and -D-S— are as defined above under pointvi);

xix) —S-D-S—S— wherein D and -D-S— are as defined above under pointvii).

Other particularly preferred compounds of the formula (Ib) are thosewherein:

b) L₂ is a linker of formula (VIII) as above defined and Z is a spacergroup which is:

xx) as defined above under point ii)

xxi) as defined above under point xv);

xxii) as defined above under point xvi);

xxiii) as defined above under point xvii);

xxiv) as defined above under point xviii);

xxv) as defined above under point xix).

Another particularly preferred object of the present invention are thecompounds of the formula (I′ a) and (I′ b):

wherein L₁, L₂, R₁ and R₂ are as defined above, Z is —NH— or a peptidicresidue constituted from 1 to 3 amino acids and Q is hydrogen atom,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, phenyl or benzyl group.

Also in this case, the compounds of the formula (I′ a) and (I′ b) can bereleased in appropriate condition giving a compound of the formula (II)as defined above.

Another particularly preferred class of compounds are the compound offormula (I) or (I′) wherein L is:

-   -   a residue of formula (IIIa) or (IVa),

wherein v and k are as defined above;

or a residue of formula (VII) or (VIII) as defined above, characterizedin that n is an integer from 2 to 5.

Linkers and Spacers

The linker L and spacer Z units attach the carrier, e.g. antibody, tothe anthracycline derivative drug moiety D through covalent bond(s). Thelinker is a bifunctional or multifunctional moiety which can be used tolink one or more drug moiety (D) and an antibody unit (Ab) to formantibody-drug conjugates (ADC) of formula Ic. The linker (L) may bestable outside a cell, i.e. extracellular, or it may be cleavable byenzymatic activity, hydrolysis, or other metabolic conditions.Antibody-drug conjugates (ADC) can be conveniently prepared using alinker having reactive functionality for binding to the drug moiety andto the antibody. A cysteine thiol, or an amine, e.g. N-terminus or aminoacid side chain such as lysine, of the antibody (Ab) can form a bondwith a functional group of a linker or spacer reagent, drug moiety (D)or drug-linker reagent (D-L).

Many positions on anthracycline derivative compounds may be useful asthe linkage position, depending upon the type of linkage. For example,ester, amide, thioamide, thiocarbamate, or carbamate linkages may beformed from the hydroxyl group of the hydroxymethyl ketone at C14; ketaland hydrazone linkages may be formed from the C₁₋₃ carbonyl group on thedrug moiety; amide, carbamate, and urea linkages may be formed from anamino group on the drug moiety D; and various alkyl, ether, thioether,disulfide, and acyl linkages may be formed from the phenyl and arylrings on the drug moiety by Friedel-Crafts type alkylation and acylationreactions.

The linkers and spacers are preferably stable extracellularly. Beforetransport or delivery into a cell, the antibody-drug conjugate (ADC) ispreferably stable and remains intact, i.e. the antibody remains linkedto the drug moiety. The linkers are stable outside the target cell andmay be cleaved at some efficacious rate inside the cell. An effectivelinker will: (i) maintain the specific binding properties of theantibody; (ii) allow intracellular delivery of the conjugate or drugmoiety; (iii) remain stable and intact, i.e. not cleaved, until theconjugate has been delivered or transported to its targetted site; and(iv) maintain a cytotoxic, cell-killing effect or a cytostatic effect ofthe anthracycline derivative drug moiety. Stability of the ADC may bemeasured by standard analytical techniques such as mass spectroscopy,HPLC, and the separation/analysis technique LC/MS.

Covalent attachment of the antibody and the drug moiety requires thelinker, and optional spacer, to have two reactive functional groups,i.e. bivalency in a reactive sense. Bivalent linker reagents which areuseful to attach two or more functional or biologically active moieties,such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, andreporter groups are known, and methods have been described theirresulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques;Academic Press: New York, p 234-242).

In another embodiment, the linker or spacer may be substituted withgroups which modulate solubility or reactivity. For example, a sulfonatesubstituent may increase water solubility of the reagent and facilitatethe coupling reaction of the linker reagent with the antibody or thedrug moiety, or facilitate the coupling reaction of Ab-L with D, or D-Lwith Ab, depending on the synthetic route employed to prepare the ADC.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). US 2007/0092940 teachesengineering antibodies by introduction of reactive cysteine amino acids.

In some embodiments, a Linker has a reactive nucleophilic group which isreactive with an electrophilic group present on an antibody. Usefulelectrophilic groups on an antibody include, but are not limited to,aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilicgroup of a Linker can react with an electrophilic group on an antibodyand form a covalent bond to an antibody unit. Useful nucleophilic groupson a Linker include, but are not limited to, hydrazide, oxime, amino,hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide. The electrophilic group on an antibody provides aconvenient site for attachment to a Linker.

Nucleophilic groups on a drug moiety include, but are not limited to:amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide groups capable of reacting toform covalent bonds with electrophilic groups on linker moieties andlinker reagents including: (i) active esters such as NHS esters, HOBtesters, haloformates, and acid halides; (ii) alkyl and benzyl halidessuch as haloacetamides; (iii) aldehydes, ketones, carboxyl, andmaleimide groups.

Spacers (Z) can be peptidic, comprising one or more amino acid units.Peptide linker reagents may be prepared by solid phase or liquid phasesynthesis methods (E. Schroder and K. Lübke, The Peptides, volume 1, pp76-136 (1965) Academic Press) that are well known in the field ofpeptide chemistry, including t-BOC chemistry (Geiser et al “Automationof solid-phase peptide synthesis” in Macromolecular Sequencing andSynthesis, Alan R. Liss, Inc., 1988, pp. 199-218) and Fmoc/HBTUchemistry (Fields, G. and Noble, R. (1990) “Solid phase peptidesynthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids”, Int. J.Peptide Protein Res. 35:161-214), on an automated synthesizer such asthe Rainin Symphony Peptide Synthesizer (Protein Technologies, Inc.,Tucson, Ariz.), or Model 433 (Applied Biosystems, Foster City, Calif.).

Exemplary amino acid linkers include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Amino acid side chains include those occurring naturally, as well asminor amino acids and non-naturally occurring amino acid analogs, suchas citrulline. Amino acid side chains include hydrogen, methyl,isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, as well as thefollowing structures:

When the amino acid side chains include other than hydrogen (glycine),the carbon atom to which the amino acid side chain is attached ischiral. Each carbon atom to which the amino acid side chain is attachedis independently in the (S) or (R) configuration, or a racemic mixture.Drug-linker reagents may thus be enantiomerically pure, racemic, ordiastereomeric.

In exemplary embodiments, amino acid side chains are selected from thoseof natural and non-natural amino acids, including alanine,2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, norleucine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,γ-aminobutyric acid, α,α-dimethyl γ-aminobutyric acid, β,β-dimethylγ-aminobutyric acid, ornithine, and citrulline (Cit).

Drug-Linker Intermediates

The invention includes drug-linker intermediates useful for conjugationto proteins, peptides, and antibodies. Such drug-linker intermediatesinclude an anthracycline derivative of formula (IIc):

Ant-L-(Z)_(m)—X  (IIc)

wherein Ant is selected from the structures:

where the wavy line indicates the attachment to L;

L is a linker selected from —N(R)—, —N(R)_(m)(C₁-C₁₂ alkylene)-,—N(R)_(m)(C₂-C₈ alkenylene)-, —N(R)_(m)(C₂-C₈ alkynylene)-,—N(R)_(m)(CH₂CH₂O)_(n)—, and the structures:

where the wavy lines indicate the attachments to Ant and Z; and

Z is an optional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂alkylene)-, and the structures:

X is a reactive functional group selected from maleimide, thiol, amino,bromide, p-toluenesulfonate, iodide, hydroxyl, carboxyl, pyridyldisulfide, and N-hydroxysuccinimide;

R is H, C₁-C₁₂ alkyl, or C₆-C₂₀ aryl;

R¹ and R² are independently selected from an amino acid side chain;

Z¹ is selected from —(C₁-C₁₂ alkylene)-, —(C₂-C₈ alkenylene)-, —(C₂-C₈alkynylene)-, and —(CH₂CH₂O)_(n)—,

m is 0 or 1; and n is 1 to 6.

Anthracycline derivatives of formula IIc include the structures:

wherein Z—X is selected from:

Exemplary drug-linker intermediate embodiments of formula IIc comprisingan anthracycline drug moiety and a linker-spacer unit include compounds50-53:

Anthracycline derivatives of formula IIc include the structure:

where Z is C₁-C₁₂ alkylene.

Exemplary drug-linker intermediate embodiments of formula IIc comprisingan anthracycline drug moiety and a linker-spacer unit include compound54 (Example 3a):

Anthracycline derivatives of formula IIc include the structures:

and where X is maleimide:

Exemplary drug-linker intermediate embodiments of formula IIc comprisingan anthracycline drug moiety and a linker-spacer unit include compound55 (Example 3b). A synthetic route to drug-linker intermediate 55 fromPNU-159682 C-14 carboxyl derivative 56 and linker-spacer intermediate 60is shown in FIG. 7 d.

Anthracycline derivatives of formula IIc include the structures:

and where Z¹ is —(C₁-C₁₂ alkylene)-.

Exemplary drug-linker intermediate embodiments of formula IIc comprisingan anthracycline drug moiety and a linker-spacer unit include thecompound:

Linker and Spacer Reagents

Beta-glucuronide linkers between the antibody and the drug moiety by aresubstrates for cleavage by beta-glucuronidase (Jeffrey et al (2006)Bioconjugate Chem. 17:831-840; WO 2007/011968). The acetal linkage ofbeta-glucuronide releases a phenolic hydroxyl on the aryl ring,potentiating “self-immolation” and 1,6-elimination of thebenzyloxycarbonyl group.

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagenthaving a maleimide stretcher and a para-aminobenzylcarbamoyl (PAB)self-immolative spacer has the structure:

where Q is C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, —NO₂ or —CN; and mis an integer ranging from 0-4.

An exemplary phe-lys(Mtr) dipeptide linker reagent having a maleimidestretcher unit and a p-aminobenzyl self-immolative Spacer unit can beprepared according to Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60, and has the structure:

where Mtr is mono-4-methoxytrityl, Q is C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),-halogen, —NO₂ or —CN; and m is an integer ranging from 0-4.

The “self-immolative linker”, PABC or PAB (para-aminobenzyloxycarbonyl),attaches the drug moiety to the antibody in the conjugate (Carl et al(1981) J. Med. Chem. 24:479-480; Chakravarty et al (1983) J. Med. Chem.26:638-644; U.S. Pat. No. 6,214,345; US20030130189; US20030096743; U.S.Pat. No. 6,759,509; US20040052793; U.S. Pat. No. 6,218,519; U.S. Pat.No. 6,835,807; U.S. Pat. No. 6,268,488; US20040018194; WO98/13059;US20040052793; U.S. Pat. No. 6,677,435; U.S. Pat. No. 5,621,002;US20040121940; WO2004/032828). Other examples of self-immolative spacersbesides PAB include, but are not limited to: (i) aromatic compounds thatare electronically similar to the PAB group such as2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med.Chem. Lett. 9:2237), thiazoles US 2005/0256030), multiple, elongated PABunits (de Groot et al (2001) J. Org. Chem. 66:8815-8830; and ortho orpara-aminobenzylacetals; and (ii) homologated styryl PAB analogs (U.S.Pat. No. 7,223,837). Spacers can be used that undergo cyclization uponamide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ringsystems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and2-aminophenylpropionic acid amides (Amsberry, et al (1990) J. Org. Chem.55:5867). Elimination of amine-containing drugs that are substituted atglycine (Kingsbury et al (1984) J. Med. Chem. 27:1447) are also examplesof self-immolative spacers useful in ADC.

Linker reagents useful for the antibody drug conjugates of the inventioninclude, but are not limited to: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and including bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, 1,8-bis-maleimidodiethyleneglycol(BM(PEO)₂), and 1,11-bis-maleimidotriethyleneglycol (BM(PEO)₃), whichare commercially available from Pierce Biotechnology, Inc.,ThermoScientific, Rockford, Ill., and other reagent suppliers.Bis-maleimide reagents allow the attachment of a free thiol group of acysteine residue of an antibody to a thiol-containing drug moiety,label, or linker intermediate, in a sequential or concurrent fashion.Other functional groups besides maleimide, which are reactive with athiol group of an antibody, anthracycline derivative drug moiety, orlinker intermediate include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

Other linker reagents are: N-succinimidyl-4-(2-pyridylthio)pentanoate(SPP), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP, Carlsson etal (1978) Biochem. J. 173:723-737),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Useful linker reagents can also beobtained via other commercial sources, such as Molecular BiosciencesInc. (Boulder, Colo.), or synthesized in accordance with proceduresdescribed in Toki et al (2002) J. Org. Chem. 67:1866-1872; U.S. Pat. No.6,214,345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO03/043583; and WO 04/032828.

The Linker may be a dendritic type linker for covalent attachment ofmore than one drug moiety through a branching, multifunctional linkermoiety to an antibody (US 2006/116422; US 2005/271615; de Groot et al(2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al (2003) Angew.Chem. Int. Ed. 42:4494-4499; Shamis et al (2004) J. Am. Chem. Soc.126:1726-1731; Sun et al (2002) Bioorganic & Medicinal Chemistry Letters12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry11:1761-1768; King et al (2002) Tetrahedron Letters 43:1987-1990).Dendritic linkers can increase the molar ratio of drug to antibody, i.e.loading, which is related to the potency of the ADC. Thus, where anantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic or branched linker.

Carrier

The carrier moiety of anthracycline derivative conjugates is derivedfrom polyclonal and monoclonal antibodies, proteins or peptides ofnatural or synthetic origin. Carrier compounds T-NH₂, T-COOH, T-CHO,T-SH, (V) or (VI) are suitable for conjugation with anthracyclinederivative compounds. Carrier moieties may be derived from polyclonalantibodies raised against tumor associated antigens; or from monoclonalantibodies binding to antigens preferentially or selectively expressedon tumor cell populations; or from natural or recombinant peptides orproteins or growth factors preferentially or selectively binding totumor cells; or from natural or synthetic polymeric carriers such aspolylysine, polyglutamic acid, polyaspartic acid and their analogues andderivatives, or such as dextran or other polymeric carbohydrateanalogues and their derivatives; or from synthetic copolymers such asthose derived from N-(2-hydroxypropyl)methacrylamide (HPMA) see: J.Kopecek, Macromolecules. H. Benoit & P. Rempp, Ed.: 505-520 (1982)Pergamon Press. Oxford, England; or from poly(aminoacid) copolymers suchas poly(GluNa, Ala, Tyr) which are useful as targetable drug-carriersfor lung tissue R. Duncan et al., Journal of Bioactive and CompatiblePolymers, Vol 4, July 1989. The carrier portion may be also derived fromportions of the above mentioned peptides or proteins obtained throughrecombinant DNA techniques.

Antibodies

Representative examples of the above mentioned antibodies and ofrespective possible therapeutic applications are: anti-T-cell antibodyT101 (Royston, I. et al., J. Immunol. 1980, 125:725); anti-CD5 antibodyOKT1 (Ortho) ATCC CRL 8000 cronic lymphocytic leukemias); anti-CD20antibody IgG1 ibritumomab, (Theuer, C. P. et al. Biotechnology AnnualReview 2004 non-hodgkin's lymphoma); anti-CD33 antibody huCD33, (Drug ofthe future 2000 25(7):686 acute myeloid leukemia); anti-transferrinreceptor antibody OKT9 (Ortho) ATCC CRL 8021 ovarian and other tumors;anti-melanoma antibody MAb 9.2.27 (Bumol, T. F. et al., Proc. Natl.Acad. Sci. USA 1982, 79:1245 melanomas); anti-carcinoma markers antibodysuch as: anti-CEA 1116 NS-3d ATCC CRL 8019), anti-alpha-fetoprotein OM3-1.1 ATCC HB 134 also hepatomas), 791T/36 (Embleton, M. J. et al., Br.J. Cancer 1981, 43, 582 also osteogenic sarcoma), B 72.3 (U.S. Pat. No.4,522,918 colorectal carcinomas and other tumors), anti-ovariancarcinoma antibody OVB 3 ATCC HB 9147, anti-breast carcinoma antibodyHMGF antigen (Aboud-Pirak, E. et al., Cancer Res. 1988, 48:3188),anti-bladder carcinoma 1G3.10 (Yu, D. S. et al., Eur. J. Urol. 1987,13:198), anti-CanAg antibody huC242 antibody (Olcher, Anthony W. et al.,Journal of Clinical Oncology 2003, 21(2):211-222 colon, pancreas,gastric), anti-prostate antibody MLN591 (Henry, Michael D. et al.,Cancer Research 2004 advanced hormone-refractory prostate cancer).

Representative examples of the above mentioned growth factors andproteins of natural or recombinant origin are FGF, EGF, PDGF, TGF-ALPHA,ALPHA -MS, Interleukins, Interferons, TNF, melanotropin (MSH), Mcm2 etc.The carrier T-CHO is preferably derived from polyclonal or monoclonalantibodies having the carbohydrate moiety, preferentially located in theFc region, selectively oxidized to aldehyde groups by means of chemicalor enzymatic methods, as described in U.S. Pat. No. 4,671,958.

The antibody unit (Ab) of Formula IIc includes any unit, type, or classof antibody that binds or reactively associates or complexes with areceptor, antigen or other receptive moiety associated with a giventarget-cell population. An antibody can be any protein or protein-likemolecule that binds to, complexes with, or reacts with a moiety of acell population sought to be therapeutically or otherwise biologicallymodified. In one aspect, the antibody unit acts to deliver theanthracycline derivative drug moiety to the particular target cellpopulation with which the antibody unit reacts. Such antibodies include,but are not limited to, large molecular weight proteins such as,full-length antibodies and antibody fragments. The antibodies of FormulaI allow attaining high concentrations of active metabolite molecules incancer cells. Intracellular targeting may be achieved by methods andcompounds which allow accumulation or retention of biologically activeagents inside cells. Such effective targeting may be applicable to avariety of therapeutic formulations and procedures.

In one embodiment, the ADC specifically binds to a receptor encoded byan ErbB gene, such as EGFR, HER2, HER3 and HER4. The ADC mayspecifically bind to the extracellular domain of the HER2 receptor. TheADC may inhibit growth of tumor cells which overexpress HER2 receptor.

In another embodiment, the antibody (Ab) of Formula IIc is a humanizedantibody such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4,huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab).

The antibodies of the invention include cysteine-engineered antibodieswhere one or more amino acids of any form of wild-type or parentantibody is replaced with a cysteine amino acid. The engineered cysteineamino acid is a free cysteine acid and not part of an intrachain orinterchain disulfide unit. Any form, type, or variant of antibody may beso engineered, i.e. mutated. For example, a parent Fab antibody fragmentmay be engineered to form a cysteine engineered Fab, referred to hereinas “ThioFab.” Similarly, a parent monoclonal antibody may be engineeredto form a “ThioMab.” It should be noted that a single site mutationyields a single engineered cysteine residue in a ThioFab, while a singlesite mutation yields two engineered cysteine residues in a ThioMab, dueto the dimeric nature of the IgG antibody. The cysteine engineeredantibodies of the invention include monoclonal antibodies, humanized orchimeric monoclonal antibodies, antigen-binding fragments of antibodies,fusion polypeptides and analogs that preferentially bind cell-associatedpolypeptides.

Cysteine-engineered antibodies have been designed as Fab antibodyfragments (thioFab) and expressed as full-length, IgG monoclonal(thioMab) antibodies (U.S. Pat. No. 7,521,541, the contents of which areincorporated by reference). ThioFab and ThioMab antibodies have beenconjugated through linkers at the newly introduced cysteine thiols withthiol-reactive linker reagents and drug-linker reagents to prepareantibody-drug conjugates (Thio ADC), including anti-HER2 (U.S. Pat. No.7,521,541), anti-CD22 (US 2008/0050310), and anti-steap1 (WO2008/052187), as well as other cysteine engineered antibodies andantibody-drug conjugates.

Antibodies comprising the antibody-drug conjugates of Formula IIcpreferably retain the antigen binding capability of their native, wildtype counterparts. Thus, antibodies of the invention are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor-associated antigens (TAA), cell surface receptorproteins and other cell surface molecules, cell survival regulatoryfactors, cell proliferation regulatory factors, molecules associatedwith (for e.g., known or suspected to contribute functionally to) tissuedevelopment or differentiation, lymphokines, cytokines, moleculesinvolved in cell cycle regulation, molecules involved in vasculogenesisand molecules associated with (for e.g., known or suspected tocontribute functionally to) angiogenesis. The tumor-associated antigenmay be a cluster differentiation factor (i.e., a CD protein). An antigento which an antibody of the invention is capable of binding may be amember of a subset of one of the above-mentioned categories, wherein theother subset(s) of said category comprise other molecules/antigens thathave a distinct characteristic (with respect to the antigen ofinterest).

In one embodiment, the antibody of antibody-drug conjugates (ADC) ofFormula IIc specifically binds to a receptor encoded by an ErbB gene.The antibody may bind specifically to an ErbB receptor selected fromEGFR, HER2, HER3 and HER4. The ADC may specifically bind to theextracellular domain (ECD) of the HER2 receptor and inhibit the growthof tumor cells which overexpress HER2 receptor. The antibody of the ADCmay be a monoclonal antibody, e.g. a murine monoclonal antibody, achimeric antibody, or a humanized antibody. A humanized antibody may behuMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,huMAb4D5-7 or huMAb4D5-8 (trastuzumab). The antibody may be an antibodyfragment, e.g. a Fab fragment.

Antibodies in Formula IIc antibody-drug conjugates (ADC) and which maybe useful in the treatment of cancer include, but are not limited to,antibodies against cell surface receptors and tumor-associated antigens(TAA). Such tumor-associated antigens are known in the art, and can beprepared for use in generating antibodies using methods and informationwhich are well known in the art. In attempts to discover effectivecellular targets for cancer diagnosis and therapy, researchers havesought to identify transmembrane or otherwise tumor-associatedpolypeptides that are specifically expressed on the surface of one ormore particular type(s) of cancer cell as compared to on one or morenormal non-cancerous cell(s). Often, such tumor-associated polypeptidesare more abundantly expressed on the surface of the cancer cells ascompared to on the surface of the non-cancerous cells. Theidentification of such tumor-associated cell surface antigenpolypeptides has given rise to the ability to specifically target cancercells for destruction via antibody-based therapies.

Examples of TAA include, but are not limited to the Tumor-AssociatedAntigens (1)-(36) listed below. For convenience, information relating tothese antigens, all of which are known in the art, is listed below andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA (1)-(36) areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

Tumor-Associated Antigens (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_(—)001203); ten Dijke, P., et al Science 264(5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); WO2004063362(Claim 2); WO2003042661 (Claim 12); U52003134790-A1 (Page 38-39);WO2002102235 (Claim 13; Page 296); WO2003055443 (Page 91-92);WO200299122 (Example 2; Page 528-530); WO2003029421 (Claim 6);WO2003024392 (Claim 2; FIG. 112); WO200298358 (Claim 1; Page 183);WO200254940 (Page 100-101); WO200259377(Page 349-350); WO200230268(Claim 27; Page 376); WO200148204 (Example; FIG. 4); NP_(—)001194 bonemorphogenetic protein receptor, type IB/pid=NP_(—)001194.1;Cross-references: MIM:603248; NP_(—)001194.1; AY065994 (2) E16 (LAT1,SLC7A5, Genbank accession no. NM_(—)003486); Biochem. Biophys. Res.Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998),Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267 (16):11267-11273);WO2004048938 (Example 2); WO2004032842 (Example IV); WO2003042661 (Claim12); WO2003016475 (Claim 1); WO200278524 (Example 2); WO200299074 (Claim19; Page 127-129); WO200286443 (Claim 27; Pages 222, 393); WO2003003906(Claim 10; Page 293); WO200264798 (Claim 33; Page 93-95); WO200014228(Claim 5; Page 133-136); US2003224454 (FIG. 3); WO2003025138 (Claim 12;Page 150); US 20050107595; US 20050106644; NP_(—)003477 solute carrierfamily 7 (cationic amino acid transporter, y+system), member5/pid=NP_(—)003477.3—Homo sapiens; Cross-references: MIM:600182;NP_(—)003477.3; NM_(—)015923; NM_(—)003486_(—)1

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_(—)012449); Cancer Res. 61 (15), 5857-5860 (2001),Hubert, R. S., et al (1999) Proc. Natl. Acad. Sci. U.S.A. 96(25):14523-14528); WO2004065577 (Claim 6); WO2004027049 (FIG. 1L);EP1394274 (Example 11); WO2004016225 (Claim 2); WO2003042661 (Claim 12);US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (FIG.2); WO200289747 (Example 5; Page 618-619); WO2003022995 (Example 9; FIG.13A, Example 53; Page 173, Example 2; FIG. 2A); NP_(—)036581 sixtransmembrane epithelial antigen of the prostate; Cross-references:MIM:604415; NP_(—)036581.1; NM_(—)012449_(—)1

(4) 0772P(CA125, MUC16, Genbank accession no. AF361486); J. Biol. Chem.276 (29):27371-27375 (2001)); WO2004045553 (Claim 14); WO200292836(Claim 6; FIG. 12); WO200283866 (Claim 15; Page 116-121); US2003124140(Example 16); US2003091580 (Claim 6); WO200206317 (Claim 6; Page400-408); Cross-references: GI:34501467; AAK74120.3; AF361486_(—)1

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_(—)005823); Yamaguchi, N., et al Biol. Chem.269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96(20):11531-11536 (1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140(1996), J. Biol. Chem. 270 (37):21984-21990 (1995)); WO2003101283 (Claim14); (WO2002102235 (Claim 13; Page 287-288); WO2002101075 (Claim 4; Page308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);Cross-references: MIM:601051; NP_(—)005814.2; NM_(—)005823_(—)1

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_(—)006424); J. Biol. Chem. 277 (22):19665-19672(2002), Genomics 62 (2):281-284 (1999), Feild, J. A., et al (1999)Biochem. Biophys. Res. Commun. 258 (3):578-582); WO2004022778 (Claim 2);EP1394274 (Example 11); WO2002102235 (Claim 13; Page 326); EP875569(Claim 1; Page 17-19); WO200157188 (Claim 20; Page 329); WO2004032842(Example IV); WO200175177 (Claim 24; Page 139-140); Cross-references:MIM:604217; NP_(—)006415.1; NM_(—)006424_(—)1

(7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878); Nagase T., et al(2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1); WO2003003984(Claim 1); WO200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Page41-43, 48-58); WO2003054152 (Claim 20); WO2003101400 (Claim 11);Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737;

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al (2002)Cancer Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180 (Claim12); US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056(Example 5); WO2003105758 (Claim 12); US2003206918 (Example 5);EP1347046 (Claim 1); WO2003025148 (Claim 20); Cross-references:GI:37182378; AAQ88991.1; AY358628_(—)1

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et alEur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79,1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et alHum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet.12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002)Hum. Genet. 111, 198-206; WO2004045516 (Claim 1); WO2004048938 (Example2); WO2004040000 (Claim 151); WO2003087768 (Claim 1); WO2003016475(Claim 1); WO2003016475 (Claim 1); WO200261087 (FIG. 1); WO2003016494(FIG. 6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Page124-125); EP522868 (Claim 8; FIG. 2); WO200177172 (Claim 1; Page297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No.5,773,223 (Claim 1a; Col 31-34); WO2004001004.

(10) MSG783 (RNF124, hypothetical protein F1120315, Genbank accessionno. NM_(—)017763); WO2003104275 (Claim 1); WO2004046342 (Example 2);WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621(Claim 1); WO2003024392 (Claim 2; FIG. 93); WO200166689 (Example 6);Cross-references: LocusID:54894; NP_(—)060233.2; NM_(—)017763_(—)1

(11) STEAP2 (HGNC_(—)8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP,prostate cancer associated gene 1, prostate cancer associated protein 1,six transmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138); Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (Claim 1; FIG. 1);WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1; FIG. 4B);WO2003104270 (Claim 11); WO2003104270 (Claim 16); US2004005598 (Claim22); WO2003042661 (Claim 12); US2003060612 (Claim 12; FIG. 10);WO200226822 (Claim 23; FIG. 2); WO200216429 (Claim 12; FIG. 10);Cross-references: GI:22655488; AAN04080.1; AF455138 1

(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_(—)017636); Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98(19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003)); US2003143557 (Claim 4); WO200040614 (Claim 14;Page 100-103); WO200210382 (Claim 1; FIG. 9A); WO2003042661 (Claim 12);WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794(Claim 14; FIG. 1A-D); Cross-references: MIM:606936; NP_(—)060106.2;NM_(—)017636_(—)1

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_(—)003203 or NM_(—)003212);Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (Claim 1); WO2003083041(Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53);WO2003024392 (Claim 2; FIG. 58); WO200216413 (Claim 1; Page 94-95, 105);WO200222808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2); Cross-references: MIM:187395;NP_(—)003203.1; NM_(—)003212_(—)1

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004); Fujisaku et al(1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al J. Exp. Med.167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad. Sci. U.S.A. 84,9194-9198, 1987; Barel M., et al Mol. Immunol. 35, 1025-1031, 1998; WeisJ. J., et al Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986; Sinha S.K., et al (1993) J. Immunol. 150, 5311-5320; WO2004045520 (Example 4);US2004005538 (Example 1); WO2003062401 (Claim 9); WO2004045520 (Example4); WO9102536 (FIG. 9.1-9.9); WO2004020595 (Claim 1); Accession: P20023;Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29,Genbank accession no. NM_(—)000626 or 11038674); Proc. Natl. Acad. Sci.U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Mulleret al (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (Claim 2,FIG. 140); WO2003087768, US2004101874 (Claim 1, page 102); WO2003062401(Claim 9); WO200278524 (Example 2); US2002150573 (Claim 5, page 15);U.S. Pat. No. 5,644,033; WO2003048202 (Claim 1, pages 306 and 309); WO99/558658, U.S. Pat. No. 6,534,482 (Claim 13, FIG. 17A/B); WO200055351(Claim 11, pages 1145-1146); Cross-references: MIM:147245;NP_(—)000617.1; NM_(—)000626_(—)1

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_(—)030764,AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci.U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem.Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (Claim 2);WO2003077836; WO200138490 (Claim 5; FIG. 18D-1-18D-2); WO2003097803(Claim 12); WO2003089624 (Claim 25); Cross-references: MIM:606509;NP_(—)110391.2; NM_(—)030764_(—)1

(17) HER2 (ErbB2, Genbank accession no. M11730); Coussens L., et alScience (1985) 230(4730):1132-1139); Yamamoto T., et al Nature 319,230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82,6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165, 869-880, 2004;Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., etal Nature 421, 756-760, 2003; Ehsani A., et al (1993) Genomics 15,426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I); WO2004009622;WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim 1);US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; FIG.1A-B); WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (Claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (Claim 52; FIG. 7); WO200020579(Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38);WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361(Claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); Accession:P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

(18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et alGenomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res.Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc. Natl. Acad. Sci.U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393 (Claim 7);WO2004044178 (Example 4); WO2004031238; WO2003042661 (Claim 12);WO200278524 (Example 2); WO200286443 (Claim 27; Page 427); WO200260317(Claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL;M18728

(19) MDP (DPEP1, Genbank accession no. BC017023); Proc. Natl. Acad. Sci.U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (Claim 1); WO200264798(Claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284 (FIG. 9);Cross-references: MIM:179780; AAH17023.1; BC017023_(—)1

(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971); Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et al Nature425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001; DumoutierL., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al J.Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003)Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (Claim 1; Page 55-59); Accession:□9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053); Gary S. C.,et al Gene 256, 139-147, 2000; Clark H. F., et al Genome Res. 13,2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A.99, 16899-16903, 2002; US2003186372 (Claim 11); US2003186373 (Claim 11);US2003119131 (Claim 1; FIG. 52); US2003119122 (Claim 1; FIG. 52);US2003119126 (Claim 1); US2003119121 (Claim 1; FIG. 52); US2003119129(Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52);US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1);

(22) EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession no.NM_(—)004442); Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061(1991) Oncogene 10 (5):897-905 (1995), Annu Rev. Neurosci. 21:309-345(1998), Int. Rev. Cytol. 196:177-244

(2000)); WO2003042661 (Claim 12); WO200053216 (Claim 1; Page 41);WO2004065576 (Claim 1); WO2004020583 (Claim 9); WO2003004529 (Page128-132); WO200053216 (Claim 1; Page 42); Cross-references: MIM:600997;NP_(—)004433.2; NM_(—)004442_(—)1

(23) ASLG659 (B7h, Genbank accession no. AX092328); US20040101899 (Claim2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3); US2003165504 (Claim1); US2003124140 (Example 2); US2003065143 (FIG. 60); WO2002102235(Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6;FIG. 10); WO200194641 (Claim 12; FIG. 7b); WO200202624 (Claim 13; FIG.1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2;Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469);WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3; Pages 190-192);WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12);WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318;

(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436); Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem.Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274(Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1);WO200281646 (Claim 1; Page 164); WO2003003906 (Claim 10; Page 288);WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b);WO200032752 (Claim 18; FIG. 1); WO9851805 (Claim 17; Page 97); WO9851824(Claim 10; Page 94); WO9840403 (Claim 2; FIG. 1B); Accession: 043653;EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGICfusion-partner-like protein/pid=AAP14954.1 Homo sapiens (human);WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example3, Claim 20); US2003194704 (Claim 45); Cross-references: GI:30102449;AAP14954.1; AY260763_(—)1

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. AF116456); BAFF receptor/pid=NP_(—)443177.1—Homosapiens; Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (Claim 35; FIG. 6B); WO2003035846 (Claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); WO200224909(Example 3; FIG. 3); Cross-references: MIM:606269; NP_(—)443177.1;NM_(—)052945 1; AF132600

(27) CD22 (B-cell receptor CD22-β isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et al (1991)J. Exp. Med. 173:137-146; WO2003072036 (Claim 1; FIG. 1);Cross-references: MIM:107266; NP_(—)001762.1; NM_(—)001771_(—)1

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation); 226 aa), pI: 4.84, MW: 25028 TM: 2[P] Gene Chromosome: 19q13.2, Genbank accession No. NP_(—)001774.10);WO2003088808, US20030228319; WO2003062401 (Claim 9); US2002150573 (Claim4, pages 13-14); WO9958658 (Claim 13, FIG. 16); WO9207574 (FIG. 1); U.S.Pat. No. 5,644,033; Ha et al (1992) J. Immunol. 148(5):1526-1531;Mueller et al (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al(1994) Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp.Immunol. 90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637;Sakaguchi et al (1988) EMBO J. 7(11):3457-3464

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa),pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accessionNo. NP_(—)001707.1); WO2004040000; WO2004015426; US2003105292 (Example2); U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1);WO200157188 (Claim 20, page 269); WO200172830 (pages 12-13); WO200022129(Example 1, pages 152-153, Example 2, pages 254-256); WO9928468 (Claim1, page 38); U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931(pages 56-58); WO9217497 (Claim 7, FIG. 5); Dobner et al (1992) Eur. J.Immunol. 22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+T lymphocytes); 273 aa, pI: 6.56MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_(—)002111.1); Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonssonet al (1989) Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol.Biol. 228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci. USA99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) TissueAntigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); US6011146(col 145-146); Kasahara et al (1989) Immunogenetics 30(1):66-68;Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No.NP_(—)002552.2); Le et al (1997) FEBS Lett. 418(1-2):195-199;WO2004047749; WO2003072035 (Claim 10); Touchman et al (2000) Genome Res.10:165-173; WO200222660 (Claim 20); WO2003093444 (Claim 1); WO2003087768(Claim 1); WO2003029277 (page 82)

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa), pI:8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No.NP_(—)001773.1); WO2004042346 (Claim 65); WO2003026493 (pages 51-52,57-58); WO200075655 (pages 106); Von Hoegen et al (1990) J. Immunol.144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci. USA99:16899-16903

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa), pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No.NP_(—)005573.1); US2002193567; WO9707198 (Claim 11, pages 39-42); Miuraet al (1996) Genomics 38(3):299-304; Miura et al (1998) Blood92:2815-2822; WO2003083047; WO9744452 (Claim 8, pages 57-61);WO200012130 (pages 24-26)

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa), pI: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.NP_(—)443170.1); WO2003077836; WO200138490 (Claim 6, FIG. 18E-1-18-E-2);Davis et al (2001) Proc. Natl. Acad. Sci. USA 98(17):9772-9777;WO2003089624 (Claim 8); EP1347046 (Claim 1); WO2003089624 (Claim 7)

(35) IRTA2 (FcRH5, Immunoglobulin superfamily receptor translocationassociated 2, a putative immunoreceptor with possible roles in B celldevelopment and lymphomagenesis; deregulation of the gene bytranslocation occurs in some B cell malignancies); 977 aa), pI: 6.88 MW:106468 TM: 1 [P] Gene Chromosome: 1q21, (Genbank accession No.Human:AF343662, AF343663, AF343664, AF343665, AF369794, AF397453,AK090423, AK090475, AL834187, AY358085; Mouse:AK089756, AY158090,AY506558; NP 112571.1); WO2003024392 (Claim 2, FIG. 97); Nakayama et al(2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836;WO200138490 (Claim 3, FIG. 18B-1-18B-2)

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBIRefSeq: NP_(—)057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;(Genbank accession No. AF179274; AY358907, CAF85723, CQ782436);WO2004074320; JP2004113151; WO2003042661; WO2003009814; EP1295944 (pages69-70); WO200230268 (page 329); WO200190304; US2004249130; US2004022727;WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579;U.S. Pat. No. 6,410,506; US 66420061; Horie et al (2000) Genomics67:146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun.266:593-602; Liang et al (2000) Cancer Res. 60:4907-12; Glynne-Jones etal (2001) Int J Cancer. Oct 15; 94(2):178-84.

Further preferred compounds are the compounds of formula (Ia), (Ib), (I′a) and (I′ b) reported as Compounds 1-14 in the Table 1 below:

TABLE 1 (I) [Ant-L—Z_(m)T (I′) Ant-L—Z—Q Comp Formula Ant L Z M Q T 1I′ IIa

—NH— — H — 2 I′ IIa

—NH— — CH₃ — 3 I′ IIa

—NH— —

— 4 I′ IIa

— H — 5 I IIa

—NH— 6 — Mcm2 6 I′ IIa

—NH— — H — 7 I′ IIa

—NH— — CH₃ — 8 I′ IIa

—NH— —

— 9 I′ IIb

— H — 10 I′ IIb

— H — 11 I IIb

NH 6 — Mcm2 12 I′ IIb

— H — 13 I IIb

S 1 — Mcm2 14 I IIa

NH 6 — Mcm2

wherein the [Ant] residue is represented by a compound of the formula(IIa) or (1%) below, that is [Ant] is a residue of an anthracycline offormula IIA as defined above,

If a chiral center or another form of an isomeric center is present in acompound of the present invention, all forms of such isomer or isomers,including enantiomers and diastereomers, are intended to be coveredherein. Compounds containing a chiral center may be used as a racemicmixture, an enantiomerically enriched mixture, or the racemic mixturemay be separated using well-known techniques and an individualenantiomer may be used alone. In cases in which compounds haveunsaturated carbon-carbon double bonds, both the cis (Z) and trans (E)isomers are within the scope of this invention.

In cases wherein compounds may exist in tautomeric forms, such asketo-enol tautomers, each tautomeric form is contemplated as beingincluded within this invention whether existing in equilibrium orpredominantly in one form.

Pharmaceutically acceptable salts of an anthracycline derivative of theformula (I′) or of a conjugate of anthracycline derivatives of theformula (I) include the acid addition salts with inorganic or organicacids, e.g., nitric, hydrochloric, hydrobromic, sulfuric, perchloric,phosphoric, acetic, trifluoroacetic, propionic, glycolic, lactic,oxalic, malonic, malic, maleic, fumaric, tartaric, citric, benzoic,cinnamic, mandelic, methanesulphonic, isethionic and salicylic acid.Preferably, the acid addition salt of the compounds of the invention isthe hydrochloride or mesylate salt.

Pharmaceutically acceptable salts of an anthracycline derivative of theformula (I′) or of a conjugate of anthracycline derivatives of theformula (I) also include the salts with inorganic or organic bases,e.g., alkali or alkaline-earth metals, especially sodium, potassium,calcium ammonium or magnesium hydroxides, carbonates or bicarbonates,acyclic or cyclic amines, preferably methylamine, ethylamine,diethylamine, triethylamine, piperidine and the like.

As used herein, unless otherwise specified, with the term C₁-C₆ alkylmeans a group such as, for instance, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl,isohexyl, and the like.

With the term C₃-C₆ cycloalkyl group means, for instance, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl, andthe like.

With the term C₁-C₆ alkylene group means a divalent residue such as, forinstance, methylene, ethylene, n-propylene, isopropylene, n-butylene,isobutylene, sec-butylene, tert-butylene, n-pentylene, neopentylene,n-hexylene, isohexylene, and the like.

The term C₃-C₆ cycloalkylene group means a divalent residue such as, forinstance, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene,cyclopentenylene, cyclohexenylene, and the like.

It is clear to the skilled man that any of the groups or substituentsherein defined may be construed from the names of the groups from whichthey originate.

As an example, unless specifically noted otherwise, in the C₁-C₅ alkoxygroup, the alkyl moiety includes, for instance, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyland the like. Exemplary C₁-C₅ alkoxy groups are methoxy (—OCH₃), ethoxy(—OCH₂CH₃), propyloxy, isopropyloxy, n-butyloxy, isobutyloxy,sec-butyloxy, tert-butyloxy, n-pentyloxy, neopentyloxy and the like.

“A peptide residue constituted from 1 to 4 amino” means a peptidecomprising a sequence from one to four natural or synthetic amino acids.

The present invention also provides processes for the preparation of acompound of the formula (I) as defined above.

A compound of the formula (Ia) as defined above and the pharmaceuticallyacceptable salts thereof may be prepared as depicted in FIG. 2.

Processes for Preparing Anthracycline Derivative Conjugates Formula (Ia)

Accordingly, a first process of the present invention for preparing acompound of the formula (Ia) as defined above and the pharmaceuticallyacceptable salts thereof, which process comprises the following steps:

Step 1 reacting a compound of formula (II) as defined above with acompound of formula (IX) or (X):

wherein v, j, k, y, and B are as defined above and R₃ is a C₁-C₃ alkylgroup;

Step 2 hydrolyzing the resultant ester intermediate (XI):

wherein R₁, R₂, R₃ are as defined above and L₁ is a group of formula(III) or (IV) as defined above;

Step 3 activating the resultant acid of formula (XII):

wherein R₁, R₂, and L₁ are as defined above and

Step 4 linking the resultant activated compound of formula (XIII):

wherein R₁, R₂, and L₁ are as defined above and W is an activating groupof the acid group, such as N-oxysuccinimido, N-oxysulfosuccinimido or2,4-dinitrophenoxy or 2,3,4,5,6-pentafluorophenoxy or t-butoxycarbonyloxy to the desired carrier so to yield compound of formula (Ia),and optionally converting the resultant compound into a pharmaceuticallyacceptable acid.

The compounds of the formula (XI), (XII) and (XIII) as defined above arealso objects of the present invention.

The reaction of step 1 is carried out in a organic solvent e.g.dimethoxyethane or preferably N,N-dimethylformamide (DMF) and in thepresence of p-toluenesulfonic acid at a temperature ranging from 0° C.to 80° C. and for a time ranging from 1 hour to 24 hours.

The reaction of step 2 was performed under basic hydrolytic conditions,preferably with a strong base like NaOH, at a temperature of from 0° C.to room temperature from a time ranging from 1 to 48 hours.

The reaction of step 3 was carried out following well known methods, forexample the N-oxysuccinimido derivative may be prepared by reaction ofthe acid (XII) with N-hydroxysuccinimide or its water soluble3-substituted sodium sulfonate salt in the presence ofN,N′-dicyclohexyl-carbodiimide in a solvent such as dichloromethane orN,N-dimethylformamide at a temperature of from 0° C. to 50° C. for atime of from 1 to 24 hours.

The reaction of step 4 can be carried out following one of methodssummarized in FIGS. 3 a, 3 b, 3 c, depending on the desired compound ofthe formula (Ia) as defined above to be obtained:

In particular, the final condensation for preparing a compound of theformula (Ia) as defined above comprises reacting a compound of formula(XIII) as define above with: Ia) a compound of formula T-[X]_(m) (XIV)wherein X is —NH₂ or —SH and m is as defined above, to obtain a compoundof the formula (Ia) as defined above under point i) or ii) respectively.

The condensation is carried out in conditions capable of creatingcovalent linkages of amidic type or thioester type and compatible withthe structure of the carrier. Preferred conditions encompass use ofbuffered aqueous solutions at pH 7-9.5, temperatures from 4° C. to 37°C., for times from some hours to several days.

For example, conditions for the condensation between the compounds offormula (XIII) and antibodies T-NH₂ are: aqueous 0.1M sodium phosphateand aqueous 0.1M sodium chloride at pH 8 containing a monoclonalantibody at 1 mg/ml, treated with a 30 fold molar excess of a 10% w/vsolution of 6 in N,N-dimethylformamide, for 24 hours at 20 (degree) C.The conjugate is purified by gel filtration on a SEPHADEX G-25 column(Pharmacia Fine Chemical, Piscataway, N.J.), eluting with PBS(phosphate-buffered saline).

Another condensation for preparing a compound of the formula (Ia) asdefined above comprises reacting a compound of formula (XIII) as defineabove with:

1b) a compound of formula (XV) NH₂-D-NH—P wherein -D- or -D-NH— are asdefined above and P is hydrogen atom or preferably a protecting group

1b′) deprotecting the NH function, if necessary, of the resultantcompound of formula (XVI):

wherein R₁, R₂, L₁ and D are as above reported and P is a protectinggroup, and then

1″b) coupling the resultant compound of the formula (XVI):

wherein R₁, R₂, L₁ and D are as above reported and P is hydrogen atom,with a carrier residue of formula T-[COOH]_(m) (XVII) wherein T and mare as defined above, so to obtain compound of formula (Ia) as definedunder the point (iii) above wherein [T-Z]— is —NH-D-NHCO-T, R₁, R₂, L₁and T are as defined above.

The reaction of step 1b is carried out in conditions capable of creatingcovalent linkages of amidic type and well known in literature andcompatible with the structure of the spacer. Preferred conditionsencompass use of buffered aqueous solutions pH 7-9.5, or organicsolvents such as, e.g. N,N-dimethylformamide, dichloromethane,tetrahydrofuran or ethyl acetate, temperature ranging from 4° C. to 50°C. and for times from some hours to several days.

The optional deprotection of step l′ b is carried out using well knownmethod reported in the literature [see, e.g. Green T. W., Wuts P. G. Min Protective Groups in Organic Chemistry]. The coupling reaction ofstep 1″b is carried out in an organic solvent, preferablyN,N-dimethylformamide in the presence of a condensing agent such as e.g.1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride1,3-di-tert-butylcarbodiimide,N-(3-dimethylaminopropyl)-n′-ethylcarbodiimide,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, orpreferably N,N′-dicyclohexyl-carbodiimide. The reaction is carried outat a temperature ranging from 5° C. to 50° C. and for a time rangingfrom 1 hour to 24 hours.

In an alternative way, the compound of formula (XVII) can be activatedwith a suitable activating acid group W as described above in step 3above and then coupled with the deprotected amine using the sameconditions reported above.

Another condensation for preparing a compound of the formula (Ia) asdefined above comprises reacting a compound of formula (XIII) as defineabove with: lc) a compound of formula (XVIII) NH₂-D-COO—P₁ wherein D orD-CO— are as defined above and P₁ is a suitable protecting acid groupe.g. alkylester that is removed after the coupling reaction to yield theacid compound of formula (XIX):

wherein R₁, R₂, L₁ and D are as described above.

The resultant compound of formula (XIX) can be used as such, orpreferably activated through a suitable activating acid group asdescribed above in step 3, and then coupled with the carrier residue offormula (XIV) T-[X]_(m) wherein X is NH₂ and m and T are as definedabove so to prepare compound of formula (Ia) defined at the point (iv)wherein [T-Z]— is —NH-D-CONH-T, R₁, R₂, L₁ and T are as defined above.

Preferred reaction conditions to couple a compound of the formula (XIII)with a compound of formula (XVIII) as defined above are the same thatreported in step 1b above. Removal of the acid protecting group iscarried out using well reviewed methods [see, e.g. Green T. W., Wuts P.G. M in Protective Groups in Organic Chemistry] for example when theacid function is protected as ethyl ester derivative the deprotectioncan be carried out under basic hydrolytic conditions preferably usingNaOH and at a temperature ranging from 0° C. to room temperature andfrom a time ranging from 1 hour to 48 hours. Reaction of a compound ofthe formula (XIX) with compound of formula (XIV) is carried out inorganic solvent preferably N,N-dimethylformamide in the presence of acondensing agent such as e.g.1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride1,3-di-tert-butylcarbodiimide,N-(3-dimethylaminopropyl)-n′-ethylcarbodiimide,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, orpreferably N,N′-dicyclohexyl-carbodiimide. The reaction is carried outat a temperature ranging from 5° C. to 50° C. and for a time rangingfrom 1 hour to 24 hours or in an alternative synthetic way activatingthe acid (XIX) with a suitable activating group as reported in step 3 ofthe process and then coupling the activated acid with a compound of theformula (XIV) under the synthetic conditions reported above in step 1.

Another condensation for preparing a compound of the formula (Ia) asdefined above comprises reacting a compound of formula (XIII) as defineabove with:

1d) a compound of the formula (XV) as described above, and then couplingthe resultant intermediate of the formula (XVI) as defined above whereinP is hydrogen atom, with a carrier residue of the formula T-[CHO]_(m)(XX), wherein m and T are as defined above, so to obtain a compound ofthe formula (Ia) described under point (v) above wherein the [T-Z]—residue represents —NH-D-N═CH-T.

Conjugation of deprotected amino derivative of formula (XVI) with acarrier of formula (XX) can be carried out in conditions capable ofcreating covalent linkages of hydrazone type and compatible with thestructure of the carrier. Preferred conditions encompass use of bufferedaqueous solutions at pH 4-7.5, alcohols or a mixture thereof, at atemperature of from 4° C. to 37° C., for times from some hours toseveral days. Conditions for the coupling between the compounds ofdeprotected derivative of formula (XVI) and antibodies T-CHO are:aqueous 0.1M sodium acetate and aqueous 0.1M sodium chloride at pH 6containing a monoclonal antibody at 1 mg/ml, treated with a 30 foldmolar excess of a 5% w/v solution of 8 in the same buffer, for 24 hoursat 20° C. The conjugate is purified by gel filtration as abovedescribed.

Another condensation for preparing a compound of the formula (Ia) asdefined above comprises reacting a compound of formula (XIII) as defineabove with:

1e) a compound of the formula (XXI) NH₂-D-SH wherein D is as definedabove, under the same reaction condition reported in step 1 of theprocess and then coupling the resultant compound of formula (XXII):

wherein R₁, R₂, L₁ and D are as reported below, either with:

1e') a carrier residue of the formula (V):

wherein T and m are as defined above, so to obtain after Michaeladdition a compound of the formula (Ia) described at the point (vi)wherein [T-Z]— is a residue of the formula (XXIII):

wherein D and T are as defined above; or

1e′) with a carrier residue of the formula (VI):

wherein T and m are as defined above, so to obtain, after displacementof the pyridine-2-thiol group, a compound of the formula (Ia) definedunder point (vii) above wherein [T-Z]— is a residue of formula (XXIV)

wherein D and T are as defined above.

The reaction of a compound of the formula (XXII) as defined above with acompound of the formula (V) as defined above can be carried out inbuffered aqueous solutions at pH 7-9.5, alcohols or a mixture thereof,at a temperature of from 4° C. to room temperature and for a period offrom 1 to 6 hours [see e.g. Willner D. et al, Bioconjugate Chem. (1993)4:521-527]. Coupling of compound (XXII) with compound (VI) is performedpreferably in a mixture of methanol and phosphate buffered solution, atpH 7.2 with from 1 to 1.5 equivalents of a compound of formula (XXII) asdefined above for each reacting group of a compound (VI) as definedabove. The reaction is incubated preferably at a temperature of from 4°C. to room temperature [see, e.g., EP328147].

A compound of the formula (Ib) as defined above and the pharmaceuticallyacceptable salts thereof may be prepared as depicted in FIG. 4.

Processes for Preparing Anthracycline Derivative Conjugates Formula (Ib)

Accordingly, the present invention provides a process of for preparing acompound of the formula (Ib) as defined above and the pharmaceuticallyacceptable salts thereof, which process comprises the following steps:

Step 1 reacting a compound of formula (II) as defined above with an acylhydrazide derivative of formula (XXV):

in conditions capable of creating covalent linkages of acyl hydrazonetype and compatible with the structure of the carrier; and

Step 2 converting the resultant compound of the formula (XXVI):

into a final compound of the formula (Ib) as defined above by anappropriate method.

The compound of the formula (XXVI) as defined above is also object ofthe present invention.

Preferred conditions of step 1 above encompass use of buffered aqueoussolutions at pH of from 4 to 7.5 or preferably an organic solvent suchas e.g. ethanol, tetrahydrofuran, or more preferably methanol, at atemperature of from 4° C. to 50° C., for a period of from 1 hour toseveral days;

The final conversion can be carried out following one of the methodssummarized in FIGS. 5 a, 5 b, 5 c:

In particular, the final condensation for preparing a compound of theformula (Ib) as defined above comprises reacting a compound of formula(XXVI) as defined above with:

2a) a carrier compound of formula T-[X]_(m), (XIV) wherein X is NH₂ orSH and m are as defined above to yield compound of formula (Ib) definedunder points (viii) and (ix) above wherein L₂ is a linker of the formula(VII) as defined above, R₁, R₂ are as defined above and [T-Z]— is T-NH—or T-S— wherein T is as defined above. Conjugation reaction is carriedout using the same conditions reported above under step 1e.

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises reacting a compound of formula (XXVI) as defineabove with:

2b) a compound of the formula (XXVII) H—R₄-D-NH—P wherein D is asdefined above, R₄ is —NH— or —S— and P is hydrogen atom or, preferably,an amino protecting group that is removed after the coupling reaction,and then

2′b) coupling the resultant acylhydrazone derivative of the formula(XXVIII):

wherein R₁, R₂, R₄ and D are as defined above, with a carrier derivativeof the formula (XVII) T-[COOH]_(m), wherein T and m are as definedabove, so to yield a compound of formula (Ib) defined at the point (x)and (xv) wherein the [T-Z]— residue is —NH-D-NHCO-T or —S-D-NHCO-T andR₁, R₂, L₂, T are as defined above. Conjugation reaction is carried outusing the same conditions reported under point 1b above.

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises reacting a compound of formula (XXVI) as defineabove with:

2c) a compound of the formula (XXIX) H—R₄-D-CO—P₁ wherein R₄, D, D-CO—and P₁ are as defined above, and removing the protecting group, ifpresent; and

2′c) coupling the resultant acylhydrazone derivative of the formula(XXX):

wherein R₁, R₂, R₄ and D are as defined above, preferably afteractivation with a suitable activating acid group W, wherein W is asreported above, with a carrier of the formula (XIV) T-[X]_(m), wherein Xis NH₂, and T and m are as defined above, so to yield a compound of theformula (Ib) as defined under points (xi) and (xvi) above wherein the[T-Z]-residue is —NH-D-CONH-T or —S-D-CONH-T and L₂, R₁, R₂, T are asdefined above. Conjugation reaction is carried out using the sameconditions reported in point (1c) of the process.

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises:

2d) coupling the compound of formula (XXVIII) obtained as describedabove with a carrier of the formula (XX) T-[CHO]_(m), so as to yield acompound of the formula (Ib) as defined under points (xii) and (xvii)above, wherein the [T-Z]— residue is —NH-D-N═C-T or —S-D-N═C-T and L₂,R₁, R₂, T are as defined above, using the same reaction conditionsreported under point 1d above;

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises reacting a compound of formula (XXVI) as defineabove with:

2e) a compound of the formula (XXXI) H—R₄-D-S—P₂ wherein R₄ and D are asdefined above and P₂ is hydrogen atom or, preferably, a thiol protectinggroup, then coupling the resultant compound of the formula (XXXII):

wherein n, R₁, R₂, R₄ and D are as defined above, after the removal ofthe thiol protecting group, if present, with either:

2e′) a carrier derivative of the formula (V) as defined above, so as toyield a compound of the formula (Ib) as defined under points (xiii) and(xviii) above wherein L₂, R₁, R₂, D, are as defined above and [T-Z]— isa residue of formula (XXIII) as defined above, or (XXIIIa) or,

2e′) a carrier derivative of the formula (VI) as defined above, so toyield compound of formula (Ib) defined under points (xiv) and (xix)above wherein L₂, R₁, R₂, D are as defined above and [T-Z]— is a residueof the formula (XXIV) as defined above or (XXIVa).

Conjugation reaction is carried out using the same conditions reportedin point (1e) of the process while removal of the selected thiolprotecting group can be carried out under the condition reported in theliterature [see, e.g. Green T. W., Wuts P. G. M in Protective Groups inOrganic Chemistry]

A compound of the formula (Ib) wherein L₂ is a spacer of formula (VIII)and the pharmaceutically acceptable salts may be obtained by a processdepicted in FIG. 6, wherein G is a carbon or nitrogen atom, preferably anitrogen atom, R₅ is halogen or hydrogen atom, preferably hydrogen atom,and n, R₁ and R₂ are as defined above.

Accordingly, the present invention provides a process for preparing acompound of the formula (Ib) as defined above wherein L₂ is a spacer offormula (VIII) and the pharmaceutically acceptable salts thereof, whichprocess comprises the following steps:

Step 1 reacting a compound of the formula (II) as defined above with anacyl hydrazide derivative of formula (XXXIII):

wherein G and R₅ are as above defined, using the same conditionsreported in step 1 as described above in the process for preparing acompound of the formula (Ib), and

Step 2 converting the resultant acylhydrazone derivative of the formula(XXXIV):

wherein n, R₁, R₂, and G are as defined above, into a final compound ofthe formula (Ib) by an appropriate method.

The compound of the formula (XXXIV) as defined above is also object ofthe present invention.

The final conversion can be carried out following one of the methodssummarized in FIGS. 7 a, 7 b, 7 c, wherein n, m, T, D, P, P₁, P₂ and R₄are as defined above. In particular, the final condensation forpreparing a compound of the formula (Ib) as defined above comprisesreacting a compound of formula (XXXIV) as defined above with:

(3a) a carrier derivative of the formula (XIV) T-[X]_(m) wherein X is—SH and m is as defined above, so to yield a compound of the formula(Ib) as defined under point (xx) above, wherein L₂ is a linker of theformula (VIII) as defined above, R₁ and R₂ are as defined above and[T-Z] is —S-T wherein T is as defined above. Reaction conditions totether the carrier to compound of formula (XXXIV) are the same describedunder point (1e′″) above.

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises reacting a compound of formula (XXXIV) as defineabove with:

(3b) a compound of formula (XXXV) SH-D-NH—P wherein D and P are asdefined above under the same conditions described under point 1e′ above,and

(3′ b) coupling the resultant acylhydrazone derivative of the formula(XXXVI), after removal of the amino protecting group, if present:

wherein n, R₁, R₂, and D are as defined above, with a carrier derivativeof the formula (XVII) T-[COOH]_(m), wherein T and m are as definedabove, so as to yield a compound of the formula (Ib) as defined underpoint (xxi) above wherein L₂ is as defined above and the [T-Z]— residueis —S-D-NHCO-T. The reaction is carried out using the same conditionsthat has been used to generate the final compounds under point (1b)above.

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises reacting a compound of formula (XXXIV) as defineabove with:

(3c) a compound of formula (XXXVII) HS-D-CO-0P₁ wherein D, and P₁ are asdefined above, under the same reaction conditions reported under point1e′ and, after removal of the amino protecting group, if present;

(3′ c) coupling the resultant acylhydrazone derivative of the formula(XXXVIII), preferably activated by reaction of a suitable activatingacid group W, wherein W is a group defined above:

wherein n, R₁, R₂, and D are as defined above, with a carrier of theformula (XIV) T-[X]_(m), wherein X is NH₂, and T and m are as definedabove, so as to yield a compound of formula (Ib) as defined under point(xxii) above wherein the [T-Z]— residue is —S-D-CONH-T and L₂, R₁, R₂, Tare as defined, using the same conditions reported under point 1c above.

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises:

(3d) coupling a compound of formula (XXXVI) obtained as described abovewith a carrier derivative of the formula (XX) T-[CHO]_(m), wherein T andm are as defined above, so as to yield a compound of the formula (Ib) asdefined under point (xxiii) above wherein the [T-Z]— residue is—S-D-N═C-T and D, L₂, R₁, R₂, T are as defined above. The reactionconditions are the same reported under point 1d above.

Another condensation for preparing a compound of the formula (Ib) asdefined above comprises reacting a compound of formula (XXXIV) asdefined above with:

(3e) a compound of the formula (XXXIX) HS-D-S—P₂ wherein D and P₂ are asdefined above, and P₂ is preferably a thiol protecting group, under thesame conditions reported under point 1e′ above, and, after removal ofthe protecting group, if present, using conditions known in theliterature and coupling the resultant acylhydrazone derivative of theformula (XL):

wherein n, R₁, R₂, and D are as defined above, with either:

(3′ e) a carrier of the formula (V) as defined above so as to yield acompound of the formula (Ib) as defined under point (xiv) above whereinL₂, R₁, R₂, D, are as defined above and [T-Z]— is a residue of formula(XXIIIa) as defined above, or

(3″e) a carrier of the formula (VI) as defined above so as to yield acompound of the formula (Ib) as defined under point (xxv) above whereinL₂, R₁, R₂ and D are as defined above and [T-Z]— is a residue of theformula (XXIVa) as defined above.

The reactions between a compound (XL) as defined above with compound offormula (VII) or (VIII) as defined above are carried out as under thesame conditions reported at the point 1e′ and 1e″ respectively.

The compounds of the present invention of the formula (I′) as definedabove, wherein L is L₁, and the pharmaceutically acceptable saltsthereof, may be obtained by a process depicted below in schemes 7-10,wherein all the symbols have the same meanings as defined above.

By reacting a compound of the formula (XIII) with a compound of theformula Q-NH₂ or Q-SH, wherein Q is as defined above, there are obtainedcompounds of formula (I′) wherein L is L₁ and R₁, R₂, Q and L₁ are asdefined above. Coupling reaction conditions are the same described aboveunder the point 1a.

By reacting a compound of the formula (XIX) with a compound of theformula Q-NH₂ as defined above there are obtained compounds of theformula (I′) wherein L is L₁ and R₁, R₂, Q and D are as defined above.Coupling reaction conditions are the same described above under thepoint 1c.

By reacting a compound of the formula (XVI) wherein P is hydrogen atomwith a compound of the formula Q-CHO, wherein Q is as defined above,there are obtained compounds of the formula (I′) wherein L is L₁ and R₁,R₂, Q, L₁ and D are as defined above. Coupling reaction conditions arethe same described above under point 1d.

By reacting a compound of the formula (XVI) wherein P is hydrogen atomand L₁ and D are as defined above, with a compound of the formula Q-COOHwherein Q is as defined above, there are obtained compounds of formula(I′) wherein L is L₁ and L₁, R₁, R₂, Q and D are as defined above.

Coupling reaction conditions are the same described above under point1b. The compounds of the present invention of the formula (I′) asdefined above, wherein L is L₂, and the pharmaceutically acceptablesalts thereof, may be obtained by a process depicted below in Schemes11-15, wherein all the symbols have the same meanings as defined above.

By reacting a compound of the formula (XXVI) with a compound of theformula Q-NH₂ or Q-SH as defined above, there are obtained compounds ofthe formula (I′) wherein L is L₂, and L₂ is of the formula (VII) asdefined above and R₁, R₂, Q are as defined above.

By reacting an acid compound of the formula (XXX) with a compound of theformula Q-NH₂ as defined above, there are obtained compounds of theformula (I′) wherein L is L₂ and L₂ is a linker of formula (VII) asdefined above and R₁, R₂, R₄, D, Q are as defined above.

By reacting an acid compound of the formula (XXVIII) with a compound ofthe formula Q-CHO as defined above, there are obtained compounds of theformula (I′) wherein L₂ is a linker of formula (VII) as defined aboveand R₁, R₂, R₄, D, Q are as defined above.

By reacting an acid compound of the formula (XXVIII) with a compound ofthe formula Q-COOH as defined above, there are obtained compounds of theformula (I′) wherein L is L₂, L₂ is a linker of the formula (VII) asdefined above and R₁, R₂, R₄, D, Q are as defined above. Couplingreaction conditions described above are the same described under point1e′.

By reacting a compound of the formula (XXXIV) with a compound of theformula Q-SH as defined above, there are obtained compounds of theformula (I′) wherein L is L₂, L₂ is a linker of the formula (VIII) asdefined above and R₁, R₂, Q are as defined above.

By reacting a compound of the formula (XXXVIII) with a compound of theformula Q-NH₂ as defined above, there are obtained compounds of theformula (I′) wherein L is L₂, L₂ is a linker of the formula (VIII) andR₁, R₂, Q and D are as defined above.

By reacting a compound of the formula (XXXVI) with a compound of theformula Q-CHO as defined above, there are obtained compounds of theformula (I′) wherein L is L₂, L₂ is a linker of the formula (VIII) andR₁, R₂, Q and D are as defined above.

By reacting a compound of the formula (XXXVI) with a compound of theformula Q-COOH as defined above, there are obtained compounds of theformula (I′) wherein L is L₂, L₂ is a linker of the formula (VIII) andR₁, R₂, Q and D are as defined above. Coupling reaction conditionsdescribed above are the same described under point 1e″above.

Starting compounds and reagent are commercially available or can beprepared following known method reported in the literature. For example,the compounds of the formula (II) are described in WO 98/02446, thecompounds of the formula (IX) and (X) are described in WO 9202255.

Antibody-Drug Conjugates

The anthracycline derivative conjugate compounds of the inventioninclude those with utility for anticancer activity. In one embodiment,the anthracycline derivative conjugate compounds include an antibodyconjugated, i.e. covalently attached by a linker, to an anthracyclinederivative drug moiety where the drug when not conjugated to an antibodyhas a cytotoxic or cytostatic effect. The biological activity of thedrug moiety is thus modulated by conjugation to an antibody.Antibody-drug conjugates (ADC) of the invention may selectively deliveran effective dose of a cytotoxic agent to tumor tissue whereby greaterselectivity, i.e. a lower efficacious dose may be achieved.

In one embodiment, the bioavailability of the ADC, or an intracellularmetabolite of the ADC, is improved in a mammal when compared to thecorresponding PNU-159682, anthracycline derivative compound alone. Also,the bioavailability of the ADC, or an intracellular metabolite of theADC is improved in a mammal when compared to the corresponding antibodyalone (antibody of the ADC, without the drug moiety or linker).

In one embodiment, the anthracycline derivative drug moiety of the ADCis not cleaved from the antibody until the antibody-drug conjugate bindsto a cell-surface receptor or enters a cell with a cell-surface receptorspecific for the antibody of the antibody-drug conjugate. The drugmoiety may be cleaved from the antibody after the antibody-drugconjugate enters the cell. The anthracycline derivative drug moiety maybe intracellularly cleaved in a mammal from the antibody of thecompound, or an intracellular metabolite of the compound, by enzymaticaction, hydrolysis, oxidation, or other mechanism.

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, G. T. (1996) BioconjugateTechniques; Academic Press: New York, p 234-242). In another embodiment,proteins containing N-terminal serine or threonine residues can reactwith sodium meta-periodate, resulting in production of an aldehyde inplace of the first amino acid (Geoghegan & Stroh, (1992) BioconjugateChem. 3:138-146; U.S. Pat. No. 5,362,852). Such aldehyde can be reactedwith a drug moiety or linker nucleophile.

In one embodiment, an antibody-drug conjugate (ADC) compound comprisesan antibody covalently attached by a linker L and an optional spacer Zto one or more anthracycline derivative drug moieties D, the compoundhaving formula (Ic)

Ab-(L-Z_(m)-D)_(p)  Ic

or a pharmaceutically acceptable salt thereof, wherein:

Ab is an antibody;

D is an anthracycline derivative selected from the structures:

where the wavy line indicates the attachment to L;

L is a linker selected from —N(R)—, —N(R)_(m)(C₁-C₁₂ alkylene)-,—N(R)_(m)(C₂-C₈ alkenylene)-, —N(R)_(m)(C₂-C₈ alkynylene)-,—N(R)_(m)(CH₂CH₂O)_(n)—, and the structures:

where the wavy lines indicate the attachments to D and Z; and

Z is an optional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂alkylene)-, and the structures:

R is H, C₁-C₁₂ alkyl, or C₆-C₂₀ aryl;

R¹ and R² are independently selected from an amino acid side chain;

Z¹ is selected from —(C₁-C₁₂ alkylene)-, —(C₂-C₈ alkenylene)-, —(C₂-C₈alkynylene)-, and —(CH₂CH₂O)_(n)—,

m is 0 or 1;

n is 1 to 6; and

p is an integer from 1 to 8.

Formula I compounds include all mixtures of variously loaded andattached antibody-drug conjugates where 1, 2, 3, 4, 5, 6, 7, and 8 drugmoieties are covalently attached to the antibody.

Exemplary embodiments of antibody-drug conjugates include:

where A_(a) is a divalent unit, such as MC (maleimidocaproyl), MP(maleimidopropanoyl) or MPEG(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)acetyl),capable of linking an antibody (Ab) to an amino acid unit, such asvaline-citrulline; and Y_(y) is a divalent unit, such as PAB(para-aminobenzyloxycarbonyl) which links an amino acid unit to the drugmoiety (D) when an amino acid unit is present. In other embodiments,A_(a) links Y_(y) directly to the drug moiety when the amino acid unitis absent. In other embodiments, the Y_(y) unit links directly the drugmoiety to the antibody unit when both the amino acid unit and the A_(a)unit are absent.

Exemplary antibody-disulfide linker drug conjugates are represented bythe structures:

The disulfide linker SPP may be constructed with linker reagentN-succinimidyl 4-(2-pyridylthio) pentanoate.

The antibody-drug conjugates of formula (I) and the compounds of theformula (I′) include all enantiomers, diastereomers, isomericallyenriched, racemic mixtures, isotopically labelled and isotopicallyenriched forms (e.g. ²H, ³H, ¹⁴C, ¹⁵N), and protected forms thereof.

Not to be limited by any particular mechanism of action, theantibody-drug conjugates of formula (I) and the compound of the formula(I′) of the present invention may be useful therapeutic agents sincethey contain an acetalic bond or a hydrazone bond, which releases theparent drug (II) upon hydronium-ion-catalyzed hydrolysis or “in vivo”enzymatic cleavage. It is well known that in malignant tumors there is ahigh rate of glycolysis compared to normal tissue, causing an increasein the production of lactate and thus a decrease of the pH in the tumorsee: H. M. Rauen et al., Z. Naturforsch, Teil B, 23 (1968) 1461. Theinvention affords a two level specificity of action of the compounds,the first one consisting in a preferential localization of the conjugatein the tumor tissue by means of antigenic recognition, and the secondone consisting in a preferential release of the drug in its active formin the tumor tissue by means of preferential acidic cleavage. While notlimiting the scope or utility of the compositions or methods of theinvention, the acid-sensitive acetal linkers described herein may becleaved in vivo under localized or systemic acidic conditions, thusseparating the targeting antibody from the drug moiety.

The conjugates produced according to the methods described arecharacterized following different chemical-physical methods. Theretention of the original molecular weight and the lack of aggregateformation may be assessed by chromatographic gel filtration procedures(Yu, D. S. et al., J. Urol. 140, 415, 1988) with simultaneous andindependent detection of anthracycline and antibody at differentwavelengths, and by gel electrophoretic methods. The overall chargedistribution of the compounds obtained may be assessed bychromatographic ion exchange methods. The anthracycline concentrationmay be assessed by spectrophotometric titration against a standardcalibration curve obtained from the parent anthracycline. The proteinconcentration may be assessed by colorimetric assays such as thebicinchonic acid assay (Smith, P. K. et al., Anal. Biochem. 150, 76,1985) or the Bradford dye assay (Bradford, M. M., (1976) Anal. Biochem.72:248). The antigen binding activity retention of the antibodies, afterthe conjugation procedures, may be assessed by an ELISA method (Yu, D.S. et al., J. Urol. 140, 415, 1988) and by cytofluorimetric methods(Gallego, J. et al., Int. J. Cancer 33, 737, 1984). The acid sensitivityof the conjugate may be evaluated by chromatographic methods afterincubation of the compounds in suitable buffered solutions.

Drug Loading

The drug loading is represented by p in an antibody-drug conjugatemolecule of Formula I, the average number of anthracycline derivativedrugs per antibody. Drug loading may range from 1 to 8 drugs (D) perantibody (Ab), i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties arecovalently attached to the antibody. Compositions of ADC of Formula Iinclude collections of antibodies conjugated with a range of drugs, from1 to 8. The average number of drugs per antibody in preparations of ADCfrom conjugation reactions may be characterized by conventional meanssuch as mass spectroscopy, ELISA assay, electrophoresis, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.By ELISA, the averaged value of p in a particular preparation of ADC maybe determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070;Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, thedistribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading, e.g. p>5, may cause aggregation,insolubility, toxicity, or loss of cellular permeability of certainantibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with thedrug-linker intermediate (D-L) or linker reagent. Only the most reactivelysine groups may react with an amine-reactive linker reagent. Also,only the most reactive cysteine thiol groups may react with athiol-reactive linker reagent. Generally, antibodies do not containmany, if any, free and reactive cysteine thiol groups which may belinked to a drug moiety. Most cysteine thiol residues in the antibodiesof the compounds exist as disulfide bridges and must be reduced with areducing agent such as dithiothreitol (DTT) or TCEP, under partial ortotal reducing conditions. Additionally, the antibody must be subjectedto denaturing conditions to reveal reactive nucleophilic groups such aslysine or cysteine. The loading (drug/antibody ratio) of an ADC may becontrolled in several different manners, including: (i) limiting themolar excess of drug-linker intermediate (D-L) or linker reagentrelative to antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(U.S. Pat. No. 7,521,541). The engineered cysteine thiols may react withlinker reagents or the drug-linker reagents of the present inventionwhich have thiol-reactive, electrophilic groups such as maleimide oralpha-halo amides to form ADC with cysteine engineered antibodies andthe anthracycline derivative drug moieties. The location of the drugmoiety can thus be designed, controlled, and known. The drug loading canbe controlled since the engineered cysteine thiol groups typically reactwith thiol-reactive linker reagents or drug-linker reagents in highyield. Engineering an IgG antibody to introduce a cysteine amino acid bysubstitution at a single site on the heavy or light chain gives two newcysteines on the symmetrical antibody. A drug loading near 2 can beachieved and near homogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

Thus the antibody-drug conjugate compositions of the invention includemixtures of antibody-drug conjugate compounds where the antibody has oneor more anthracycline derivative drug moieties and where the drugmoieties may be attached to the antibody at various amino acid residues.

Preparation of Antibody-Drug Conjugates

The ADC of Formula I may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a nucleophilic group oran electrophilic group of an antibody with a bivalent linker reagent, toform antibody-linker intermediate Ab-L, via a covalent bond, followed byreaction with an activated drug moiety reagent; and (2) reaction of anucleophilic group or an electrophilic group of a drug moiety reagentwith a linker reagent, to form drug-linker reagent D-L, via a covalentbond, followed by reaction with the nucleophilic group or anelectrophilic group of an antibody. Conjugation methods (1) and (2) maybe employed with a variety of antibodies, drug moieties, and linkers toprepare the antibody-drug conjugates of Formula I.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.). Each cysteinedisulfide bridge will thus form, theoretically, two reactive thiolnucleophiles. Additional nucleophilic groups can be introduced intoantibodies through the reaction of lysines with 2-iminothiolane (Traut'sreagent) resulting in conversion of an amine into a thiol.

Antibody-drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, G. T. (1996) BioconjugateTechniques; Academic Press: New York, p 234-242). In another embodiment,proteins containing N-terminal serine or threonine residues can reactwith sodium meta-periodate, resulting in production of an aldehyde inplace of the first amino acid (Geoghegan & Stroh, (1992) BioconjugateChem. 3:138-146; U.S. Pat. No. 5,362,852). Such aldehyde can be reactedwith a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups. Reactive nucleophilic groups may beintroduced on the anthracycline derivative compounds by standardfunctional group interconversions. For example, hydroxyl groups may beconverted to thiol groups by Mitsunobu-type reactions, to formthiol-modified drug compounds.

The antibody-drug conjugates in Table 2 were prepared according to thedescribed methods in the Examples and tested for efficacy by in vitrocell proliferation assay and in vivo tumor xenograft growth inhibition.

TABLE 2 Antibody-drug conjugates linker- No. ADC formula Figures drugDAR * 101 Tr-MCC-DM1 12-20, 22, SMCC-DM1 3.4 24, 26, 28, 31 102thio-HC-Tr- 12-15, 22, 51 2.18 maleimide-ketal-Ant 24, 26, 28, 30, 31103 thio-HC-Tr- 12-15, 20, 52 2.4 maleimide-hydrazone-Ant 22, 24, 26,28, 30 104 thio- HC-Tr- 16-20, 22, 53 1.25 thiopyridine-hydrazone-Ant24, 26, 28 105 thio-HC-Tr- 16-20, 22, 50 1.6 NHS-ketal-Ant 24, 26, 28106 thio-HC-Tr- 20, 22, 24, MC-vc- 1.9 MC-vc-PAB-MMAE 26, 28 PAB-MMAE107 thio- HC-anti-CD22- 21, 23, 25, 51 2.57 maleimide-ketal-Ant 27, 29,30, 31, 32 108 thio-HC-anti-CD22- 21, 23, 25, 52 2.43 maleimidehydrazone-Ant 27, 29, 30 109 thio-HC-anti-CD22- 21, 23, 25, 53 1.43thiopyridine-hydrazone-Ant 27, 29, 110 anti-CD22-NHS-ketal-Ant 16-19,21, 50 2.13 23, 25, 27, 29, 111 thio-HC-anti-CD22- 30 MC-vc- 1.94MC-vc-PAB-MMAE PAB-MMAE 112 thio-HC-anti-steap1- 32 MC-vc- 2MC-vc-PAB-MMAE PAB-MMAE 113 thio-HC-anti-steap1- 32 51 1.65maleimide-ketal-Ant * DAR = drug/antibody ratio average

Screening for Antibody-Drug Conjugates (ADC) Directed AgainstTumor-Associated Antigens and Cell Surface Receptors

Assay methods for detecting cancer cells comprise exposing cells to anantibody-drug conjugate compound, and determining the extent of bindingof the antibody-drug conjugate compound to the cells. Formula I ADCcompounds which are identified in the animal models and cell-basedassays can be further tested in tumor-bearing higher primates and humanclinical trials.

Transgenic animals and cell lines are particularly useful in screeningantibody-drug conjugates (ADC) that have potential as prophylactic ortherapeutic treatments of diseases or disorders involving overexpressionof tumor-associated antigens and cell surface receptors, e.g. HER2 (U.S.Pat. No. 6,632,979). Screening for a useful ADC may involveadministering candidate ADC over a range of doses to the transgenicanimal, and assaying at various time points for the effect(s) of the ADCon the disease or disorder being evaluated. Alternatively, oradditionally, the drug can be administered prior to or simultaneouslywith exposure to an inducer of the disease, if applicable. Candidate ADCmay be screened serially and individually, or in parallel under mediumor high-throughput screening format. The rate at which ADC may bescreened for utility for prophylactic or therapeutic treatments ofdiseases or disorders is limited only by the rate of synthesis orscreening methodology, including detecting/measuring/analysis of data.

One embodiment is a screening method comprising (a) transplanting cellsfrom a stable breast cancer cell line into a non-human animal, (b)administering an ADC drug candidate to the non-human animal and (c)determining the ability of the candidate to inhibit the formation oftumors from the transplanted cell line. The invention also concerns amethod of screening ADC candidates for the treatment of a disease ordisorder characterized by the overexpression of a receptor proteincomprising (a) contacting cells from a stable breast cancer cell linewith a drug candidate and (b) evaluating the ability of the ADCcandidate to inhibit the growth of the stable cell line.

One embodiment is a screening method comprising (a) contacting cellsfrom a stable breast cancer cell line with an ADC drug candidate and (b)evaluating the ability of the ADC candidate to induce cell death, induceapoptosis, block heregulin binding, block ligand-stimulated tyrosinephosphorylation, or block ligand activation of HER2. Another embodimentthe ability of the ADC candidate to is evaluated. In another embodimentthe ability of the ADC candidate to is evaluated.

Another embodiment is a screening method comprising (a) administering anADC drug candidate to a transgenic non-human mammal that overexpresses,e.g. in its mammary gland cells, a native human protein, e.g. HER2 or afragment thereof, wherein such transgenic mammal has stably integratedinto its genome a nucleic acid sequence encoding the native humanprotein or a fragment thereof having the biological activity of thenative human protein, operably linked to transcriptional regulatorysequences directing its expression, and develops a tumor. Candidate ADCare screened by being administered to the transgenic animal over a rangeof doses, and evaluating the animal's physiological response to thecompounds over time. Administration may be oral, or by suitableinjection, depending on the chemical nature of the compound beingevaluated. In some cases, it may be appropriate to administer thecompound in conjunction with co-factors that would enhance the efficacyof the compound. If cell lines derived from the subject transgenicanimals are used to screen for compounds useful in treating variousdisorders associated with overexpression of certain tumor-associatedantigen proteins or cell surface receptors, e.g. HER2-overexpression. Toidentify growth inhibitory ADC compounds that specifically target HER2,one may screen for ADC which inhibit the growth of HER2-overexpressingcancer cells derived from transgenic animals (U.S. Pat. No. 5,677,171)

In Vitro Cell Proliferation Assay

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells havingtumor-associated antigens or receptor proteins to the antibody of theADC in a cell culture medium; culturing the cells for a period fromabout 6 hours to about 5 days; and measuring cell viability. Cell-basedin vitro assays may be used to measure viability, i.e. proliferation(IC₅₀), cytotoxicity (EC₅₀), and induction of apoptosis (caspaseactivation) of the ADC. The CellTiter-Glo® Luminescent Cell ViabilityAssay is a commercially available (Promega Corp., Madison, Wis.),homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670). This cell proliferation assay determines thenumber of viable cells in culture based on quantitation of the ATPpresent, an indicator of metabolically active cells (Crouch et al (1993)J. Immunol. Meth. 160:81-88; U.S. Pat. No. 6,602,677). TheCellTiter-Glo® Assay is conducted in 96 well format, making it amenableto automated high-throughput screening (HTS) (Cree et al (1995)AntiCancer Drugs 6:398-404). The homogeneous assay procedure involvesadding the single reagent (CellTiter-Glo® Reagent) directly to cellscultured in serum-supplemented medium. Cell washing, removal of mediumand multiple pipetting steps are not required. The system detects as fewas 15 cells/well in a well format in 10 minutes after adding reagent andmixing.

The evaluation of the retention of cytotoxicity of conjugates incomparison with the parent drug may be assessed by a test based on thequantification of ATP. A2780 human ovarian and MCF7 human breast cancercells (1250 cells/well) were seeded in white 384 well-plates in completemedium (RPMI1640 or EMEM plus 10% Fetal bovine serum) and treated withcompounds dissolved in 0.1% DMSO, 24h after seeding. The cells wereincubated at 37° C. and 5% CO2 and after 72 hours the plates wereprocessed using CellTiter-Glo assay (Promega) following themanufacturer's instruction.

CellTiter-Glo is a homogenous method based on the quantification of theATP present, an indicator of metabolically active cells. ATP isquantified using a system based on luciferase and D-luciferin resultinginto light generation. The luminescent signal is proportional to thenumber of cells present in culture. Briefly, 25 μL/well reagentsolutions were added to each well and after 5 minutes shackingmicroplates were read by a luminometer. The luminescent signal isproportional to the number of cells present in culture.

The anti-proliferative effects of antibody-drug conjugates of Formula Ic(Table 6) were measured by the CellTiter-Glo® Assay (Example 9) againstthe HER2 expressing tumor cell lines in FIGS. 8-29 in 3 day continuousexposure studies.

FIG. 8 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: free drug PNU-159682 continuous exposure, PNU-1596821 hr incubation, linker drug: NHS-ketal-Ant 50, linker drug:maleimide-ketal-Ant 51, linker drug: maleimide-hydrazone-Ant 52, andlinker drug: thiopyridine-hydrazone-Ant 53. The HER2 expression level ofSK-BR-3 cells is 3+. SK-BR-3 cell proliferation was most potentlyinhibited by continuous exposure to PNU-159682. Brief (1 hr) exposurealso effectively inhibited growth of SK-BR-3 cells. Hydrazone-linked Ant52 and 53 were less potent than free Ant, while ketal-linked Ant 50 and51 linker drug intermediates showed minimal anti-proliferative activity.

FIG. 9 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: free drug: PNU-159682 continuous exposure, free drug:PNU-159682 1 hr incubation, linker drug: NHS-ketal-Ant 50, linker drug:maleimide-ketal-Ant 51, linker drug: maleimide-hydrazone-Ant 52, andlinker drug: thiopyridine-hydrazone-Ant 53. The HER2 expression level ofBT-474 cells is 3+. BT-474 cell proliferation was most potentlyinhibited by continuous exposure to PNU-159682. Brief (1 hr) exposurealso effectively inhibited growth of BT-474 cells. Hydrazone-linked Ant52 and 53 showed minimal anti-proliferative activity, while ketal-linkedAnt 50 and 51 were inactive.

FIG. 10 shows a plot of MCF7 in vitro cell viability at 3 days versusconcentrations of: free drug: PNU-159682 continuous exposure, free drug:PNU-159682 1 hr incubation, linker drug: NHS-ketal-Ant 50, linker drug:maleimide-ketal-Ant 51, linker drug: maleimide-hydrazone-Ant 52, andlinker drug: thiopyridine-hydrazone-Ant 53. MCF7 cell proliferation wasmost potently inhibited by continuous exposure to PNU-159682. Brief (1hr) exposure also effectively inhibited growth of MCF7 cells.Hydrazone-linked Ant 52 and 53 showed minial anti-proliferativeactivity, while ketal-linked Ant 50 and 51 were inactive.

FIG. 11 shows a plot of doxorubicin-resistant (DoxRes) HER2 in vitrocell viability at 3 days versus concentrations of: free drug: PNU-159682continuous exposure, free drug: PNU-159682 1 hr incubation, linker drug:NHS-ketal-Ant 50, linker drug: maleimide-ketal-Ant 51, linker drug:maleimide-hydrazone-Ant 52, and linker drug: thiopyridine-hydrazone-Ant53. The HER2 expression level of DoxRes HER2 cells is 3+, with highPgP/MDR1. DoxRes/HER2 cell proliferation was most potently inhibited bycontinuous exposure to PNU-159682. Brief (1 hr) exposure alsoeffectively inhibited growth of AdrRes/HER2 cells. Hydrazone-linked Ant52 and 53 were less potent than free Ant, while ketal-linked Ant 50 and51 showed minimal anti-proliferative activity. The DoxRes Her2 cell lineis also known as “AdrRes Her2”.

FIG. 12 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102, andthio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103.Thio-Tr-HC-maleimide-hydrazone-Ant 103 showed the most potentanti-proliferative activity on SK-BR-3 cells.Thio-Tr-HC-maleimide-ketal-Ant 102 and Tr-MCC-DM1 101 were equallypotent in terms of IC₅₀, but treatment of SK-BR-3 cells with 102resulted in greater cell killing than 101. All conjugates tested weremore potent than trastuzumab.

FIG. 13 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102, andthio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103.Thio-Tr-HC-maleimide hydrazone-Ant 103 showed the most potentanti-proliferative activity on BT-474 cells.Thio-Tr-HC-maleimide-ketal-Ant 102 and Tr-MCC-DM1 101 were equallypotent in inhibiting growth of BT-474 cells. All conjugates tested weremore potent than trastuzumab.

FIG. 14 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, trastuzumab-MCC-DM1 101,thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102, andthio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103. The HER2expression level of MCF7 cells is normal.Thio-Tr-HC-maleimide-hydrazone-Ant 103 showed potent anti-proliferativeactivity on the Her2-normal MCF7 cell line. Trastuzumab, Tr-MCC-DM1 101and thio-Tr-HC-maleimide-ketal-Ant 102 were not active on MCF7 cells.

FIG. 15 shows a plot of doxorubicin-resistant (DoxRes) HER2 in vitrocell viability at 3 days versus concentrations of: trastuzumab,trastuzumab-MCC-DM1 101, thio-trastuzumab (HC A114C)-maleimide ketal-Ant102, and thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103.Thio-Tr-HC-maleimide-hydrazone-Ant 103 showed potent anti-proliferativeactivity on DoxRes/HER2 cells, which express high levels of both HER2and the multi-drug resistance transporter MDR1/PgP.Thio-Tr-HC-maleimide-ketal-Ant 103 was active only at the two highestconcentrations tested (3.3 and 10 ug/ml) whereas Tr-MCC-DM1 101 andtrastuzumab showed minimal activity on this cell line.

FIG. 16 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: anti-CD22 NHS ketal-Ant 110, trastuzumab,trastuzumab-MCC-DM1 101, and thio-trastuzumab (HC A114C)-NHS-ketal-Ant105. Thio-Tr-HC-NHS-ketal-Ant 105 and Tr-MCC-DM1 101 showed equivalentpotency on SK-BR-3 cell proliferation in terms of IC₅₀; with 105treatment resulting in greater cell killing. The non-targeted controlanti-CD22-NHS-ketal-Ant 110 also showed potent anti-proliferativeactivity on SK-BR-3 cells. All conjugates tested were more potent thantrastuzumab.

FIG. 17 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: anti-CD22 NHS ketal-Ant 110, trastuzumab,trastuzumab-MCC-DM1 101, and thio-trastuzumab (HC A114C)-NHS-ketal-Ant105. Thio-Tr-HC-NHS ketal-Ant 105 showed the most potentanti-proliferative activity on BT-474 cells. Treatment with Tr-MCC-DM1also resulted in growth inhibition of BT-474 cells. The non-targetedcontrol anti-CD22-NHS-ketal-Ant 110 showed potent anti-proliferativeeffects on BT-474 cells. All conjugates tested were more potent thantrastuzumab.

FIG. 18 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: anti-CD22 NHS ketal-Ant 110, trastuzumab,trastuzumab-MCC-DM1 101, and thio-trastuzumab (HC A114C)—NHS-ketal-Ant105. Thio-Tr-HC-NHS-ketal-Ant 105 and anti-CD22-NHS-ketal-Ant 110 showedequivalent activity on low HER2-expressing MCF7 cells. Trastuzumab andTr-MCC-DM1 101 were not active on MCF7.

FIG. 19 shows a plot of doxorubicin-resistant (DoxRes) Her2 in vitrocell viability at 3 days versus concentrations of: anti-CD22 NHSketal-Ant 110, trastuzumab, trastuzumab-MCC-DM1 101, andthio-trastuzumab (HC A114C)-NHS-ketal-Ant 105. Thio-Tr-HC-NHS-ketal-Ant105 and anti-CD22-NHS-ketal-Ant 110 showed equivalent activity onDoxRes/HER2 cells. Tr-MCC-DM1 101 showed modest activity, andtrastuzumab had no effect on DoxRes/HER2.

FIG. 20 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102,thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103,thio-trastuzumab (HC A114C)-thiopyridine hydrazone-Ant 104,thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105, trastuzumab-MCC-DM1 101,and thio-trastuzumab (HC A 114C)-MC-vc-PAB-MMAE 106.

FIG. 21 shows a plot of SK-BR-3 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, thio-anti-CD22 (HC A114C)-maleimideketal-Ant 107, thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108,thio-anti-CD22 (HC A114C)-thiopyridine hydrazone-Ant 109,anti-CD22-NHS-ketal-Ant 110, and PNU-159682 free drug. As previouslyreported, trastuzumab modestly inhibits the growth of SK-BR-3 cellsthrough a cytostatic mechanism. Non-targeted control ADCsthio-anti-CD22-HC-ketal-Ant 107 and anti-CD22-NHS-ketal-Ant 110 showedanti-proliferative activity only at the highest doses tested.Non-targeted control ADCs thio-anti-CD22-HC-maleimide hydrazone-Ant 108and thio-anti-CD22-H-thiopyridine-hydrazone-Ant 109 showed equivalentpotency for inhibiting SK-BR-3 cell growth as the HER2-targeted ADCs(FIG. 20), indicating lability of hydrazone-linked ADCs. Free drugPNU-159682, administered in nM concentrations, caused cytotoxicity atall doses tested.

FIG. 22 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102,thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103,thio-trastuzumab (HC A114C)-thiopyridine hydrazone-Ant 104,thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105, trastuzumab-MCC-DM1 101,and thio-trastuzumab (HC A114C)-MC-vc-PAB-MMAE 106. All ADCs testedshowed similar activity on BT-474 cell proliferation. Thio-Tr-HC-vc-MMAE106 had the lowest IC₅₀ (0.01 ng/ml), while treatment withthio-Tr-HC-maleimide-hydrazone-Ant 103 andthio-Tr-HC-thiopyridine-hydrazone-Ant 104 resulted in the greatestamount of total growth inhibition.

FIG. 23 shows a plot of BT-474 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, thio-anti-CD22 (HC A114C)-maleimideketal-Ant 107, thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108,thio-anti-CD22 (HC A114C)-thiopyridine hydrazone-Ant 109,anti-CD22-NHS-ketal-Ant 110, and PNU-159682 free drug. As previouslyreported, trastuzumab modestly inhibits the growth of BT-474 cellsthrough a cytostatic mechanism. Non-targeted control ADCsthio-anti-CD22-HC-ketal-Ant 107 and anti-CD22-NHS-ketal-Ant 110 had noanti-proliferative effect on BT-474 cells. Non-targeted control ADCsthio-anti-CD22-HC-maleimide hydrazone-Ant 108 andthio-anti-CD22-H-thiopyridine-hydrazone-Ant 109 showed equivalentpotency for inhibiting BT-474 cell growth as the HER2-targeted ADCs(FIG. 22), indicating lability of hydrazone-linked ADCs. Free drugPNU-159682, administered in μM concentrations, caused cytotoxicity atall doses tested.

FIG. 24 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102,thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103,thio-trastuzumab (HC A114C)-thiopyridine hydrazone-Ant 104,thio-trastuzumab (HC A 114C)-NHS-ketal-Ant 105, trastuzumab-MCC-DM1 101,and thio-trastuzumab (HC A114C)-MC-vc-PAB-MMAE 106. Onlythio-Tr-HC-maleimide-hydrazone-Ant 103 and thio-Tr-HC-thiopyridine-Ant104 had anti-proliferative effects on HER2-low MCF7 cells, indicatinglability of hydrazone-linked ADCs. All other ADCs tested(thio-Tr-HC-vc-MMAE 106, Tr-MCC-DM1 101, thio-Tr-HC-maleimide-ketal-Ant102 and thio-Tr-NHS-ketal-Ant 105) had no effect on MCF7 cell growth.

FIG. 25 shows a plot of MCF-7 in vitro cell viability at 3 days versusconcentrations of: trastuzumab, thio-anti-CD22 (HC A114C)-maleimideketal-Ant 107, thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108,thio-anti-CD22 (HC A114C)-thiopyridine hydrazone-Ant 109,anti-CD22-NHS-ketal-Ant 110, and PNU-159682 free drug. Consistent withprevious reports, trastuzumab was completely inactive on lowHER2-expressing MCF7 cells. Non-targeted control ADCsthio-anti-CD22-HC-maleimide-ketal-Ant 107 andthio-anti-CD22-NHS-ketal-Ant 110 also did not inhibit growth of MCF7cells, indicating lack of drug release from stable ketal linkers.Hydrazone-linked non-targeted control ADCs 108 and 109 showedanti-proliferative effects on MCF7 cells, indicating release of drugfrom labile hydrazone-linked ADCs. Free drug PNU-159682 administered innM doses, caused cytotoxicity at all concentrations tested.

FIG. 26 shows a plot of doxorubicin-resistant (DoxRes)/HER2 in vitrocell viability at 3 days versus concentrations of: thio-trastuzumab (HCA114C)-maleimide ketal-Ant 102, thio-trastuzumab (HC A114C)-maleimidehydrazone-Ant 103, thio-trastuzumab (HC A114C)-thiopyridinehydrazone-Ant 104, thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105,trastuzumab-MCC-DM1 101, and thio-trastuzumab (HC A 114C)-MC-vc-PAB-MMAE106. While 106 had no effect and Tr-MCC-DM1 101 had minimal effect ongrowth of DoxRes/HER2 cells, ketal-linked ADC 102 and 105 were activeonly at the highest concentrations tested, while hydrazone-linked ADC103 and 104 potently inhibited growth of DoxRes/HER2 cells

FIG. 27 shows a plot of doxorubicin-resistant (DoxRes)/HER2 in vitrocell viability at 3 days versus concentrations of: trastuzumab,thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107, thio-anti-CD22 (HCA114C)-maleimide hydrazone-Ant 108, thio-anti-CD22 (HC A114C)-thiopyridine hydrazone-Ant 109, anti-CD22-NHS-ketal-Ant 110, andPNU-159682 free drug. Trastuzumab alone had no effect on growth ofDoxRes/HER2 cells. Ketal-linked non-targeted control ADC 107 and 110inhibited growth of DoxRes/HER2 cells only at the highest concentrationstested, while hydrazone-linked non-targeted control ADC 108 and 109showed potent anti-proliferative activity on DoxRes/HER2 cells.Activities of all four non-targeted anti-CD22-Ant control ADC weresimilar to HER2-targeted thio-Ant ADC (FIG. 26). Free drug PNU-159682,administered in μM doses, inhibited growth at all concentrations tested.

FIG. 28 shows a plot of doxorubicin-resistant (DoxRes)/HER2 in vitrocell viability at 3 days versus concentrations of: thio-trastuzumab (HCA114C)-maleimide ketal-Ant 102, thio-trastuzumab (HC A114C)-maleimidehydrazone-Ant 103, thio-trastuzumab (HC A114C)-thiopyridinehydrazone-Ant 104, thio-trastuzumab (HC A114C)-NHS-ketal-Ant 105,trastuzumab-MCC-DM1 101, and thio-trastuzumab (HC A 114C)-MC-vc-PAB-MMAE106, all administered in the presence of verapamil (10 μg/m). Verapamilinhibits efflux of diverse drugs known to be substrates of themulti-drug resistance transporter MDR1/P-glycoprotein (PgP), which ishighly expressed on DoxRes/HER2 cells. Addition of verapamil renderedthe DoxRes/HER2 cells sensitive to the cytotoxic effects of Tr-MCC-DM1101, indicating that DM1 is a substrate for MDR1/PgP. Verapamil had noeffect on the response to the other ADCs tested (102-106), indicatingthat the drug effects of these ADCs are not inhibited by NDR1/PgP.

FIG. 29 shows a plot of doxorubicin-resistant (DoxRes)/HER2 in vitrocell viability at 3 days versus concentrations of: trastuzumab,thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107, thio-anti-CD22 (HCA114C)-maleimide hydrazone-Ant 108, thio-anti-CD22 (HCA114C)-thiopyridine hydrazone-Ant 109, anti-CD22-NHS-ketal-Ant 110, andPNU-159682 free drug, all administered in the presence of verapamil. AllADCs tested (107-110) were equally active in the presence of verapamil(compare to FIG. 27), indicating that the Ant drug is not a substrate ofMDR1/PgP.

Table 3 summarizes the IC₅₀ values (ug/ml) for inhibition of SK-BR-3,BT-474, MCF7, doxorubicin-resistant (DoxRes), and doxorubicin-resistant(DoxRes) with verapamil cell proliferation of the test compounds ofFIGS. 20-29. Comparing SK-BR-3 and BT-474 to MCF7, themaleimide-ketal-Ant and NHS-ketal-Ant ADC show target-dependent killingwhereas hydrazone-linked-Ant ADC show target-independent, non-selectivekilling.

TABLE 3 in vitro inhibition of SK-BR-3, BT-474, MCF7,doxorubicin-resistant (DoxRes), and doxorubicin-resistant (DoxRes) withverapamil cell proliferation IC50 (ug/ml) IC50 (ug/ml) IC50 (ug/ml) IC50(ug/ml) IC50 (ug/ml) Adr-res HER2 + SK-BR-3 BT-474 MCF7 Adr-res HER2verapamil Test compound FIGS. 20, 21 FIGS. 22, 23 FIGS. 24, 25 FIGS. 26,27 FIGS. 28, 29 thio-trastuzumab (HC 0.0014 0.0341 — 1.5 0.284A114C)-maleimide ketal-Ant 102 thio-trastuzumab (HC 0.0022 0.032  0.11020.005 0.005 A114C)-maleimide hydrazone-Ant 103 thio-trastuzumab (HC0.005 0.0897 0.1390 0.009 0.014 A114C)-thiopyridine hydrazone-Ant 104thio-trastuzumab (HC 0.014 0.0369 — 5.662 0.4 A114C)-NHS-ketal-Ant 105trastuzumab-MCC-DM1 101 0.0032 0.0287 — — 0.021 thio-trastuzumab (HC0.0036 0.0075 — — — A114C)-MC-vc-PAB-MMAE 106 trastuzumab — — — — —thio-anti-CD22 (HC A114C)- 1.588 — — 4.827 2.541 maleimide ketal-Ant 107thio-anti-CD22 (HC A114C)- 0.0025 0.049  0.0437 0.005 0.006 maleimidehydrazone-Ant 108 thio-anti-CD22 (HC A114C)- 0.011 0.1331 0.1367 0.0150.015 thiopyridine hydrazone-Ant 109 anti-CD22-NHS-ketal-Ant 110 1.615 —— 3.254 1.685 PNU-159682 free drug — — — — —

The anti-proliferative effects of antibody-drug conjugates of Formula Ic(Table 6) were measured by the CellTiter-Glo® Assay (Example 9) againstCD22 positive, B-lymphoma cell lines: BJAB, GRANTA, DoHH2 and SuDHL4 in3 day continuous exposure studies. Jurkat cells (CD22 negative) weretreated with the antibody-drug conjugates as a negative control.

Table 4 summarizes the IC₅₀ values (ug/ml) for inhibition of BJAB,GRANTA, DoHH2 SuDHL4 and Jurkat cell lines by test antibody-drugconjugate compounds in 3 day continuous exposure studies. The anti-CD22antibody-drug conjugates 107-110 showed highly potent cell killingeffects. Significant cell killing was observed with negative controlanti-HER2 antibody drug conjugates 102-105. Significant cell killing wasobserved with anti-CD22 antibody-drug conjugates 107-110 on CD22negative Jurkat cells.

TABLE 4 in vitro inhibition of BJAB, GRANTA, DoHH2 SuDHL4 and Jurkatcell lines IC50 (ug/ml) IC50 (ug/ml) IC50 (ug/ml) IC50 (ug/ml) IC50(ug/ml) test ADC BJAB GRANTA DoHH2 SuDHL4 JURKAT thio-trastuzumab (HC0.9863 0.3318 0.2053 0.7990 2.2985 A114C)-maleimide ketal-Ant 102thio-trastuzumab (HC <1.28 × 10⁻⁶ <1.28 × 10⁻⁶ <1.28 × 10⁻⁶ <1.28 × 10⁻⁶2.09 × 10⁻⁵ A114C)-maleimide hydrazone-Ant 103 thio-trastuzumab(HC >10 >10 >10 >10 >10 A114C)-thiopyridine hydrazone-Ant 104thio-trastuzumab (HC 0.01197 0.00265 0.00189 0.00966 0.02811A114C)-NHS-ketal- Ant 105 thio-anti-CD22 (HC 0.000312 <1.28 × 10⁻⁶ <1.28× 10⁻⁶ <1.28 × 10⁻⁶ 0.001446 A114C)-maleimide ketal-Ant 107thio-anti-CD22 (HC <1.28 × 10⁻⁶ <1.28 × 10⁻⁶ <1.28 × 10⁻⁶ <1.28 × 10⁻⁶4.60 × 10⁻⁵ A114C)-maleimide hydrazone-Ant 108 thio-anti-CD22 (HC   9.83× 10⁻⁵ <1.28 × 10⁻⁶ <1.28 × 10⁻⁶ 0.000105 0.000412 A114C)-thiopyridinehydrazone-Ant 109 anti-CD22-NHS-ketal- 0.001217 0.000219 0.0001250.001066 0.004702 Ant 110

In Vivo Serum Clearance and Stability in Mice

Serum clearance and stability of ADC may be investigated in nude, naive(without tumors received by exogenous grafts) mice according to theprocedures in Example 10. A difference in the amount of total antibodyand ADC indicates cleavage of the linker and separation of the antibodyfrom its drug moiety.

Stability of the conjugates was studied using HPLC analysis. Theconjugate was incubated in ammonium acetate buffers at pH 4 and 5.2 at37° C. Each solution was then taken periodically and applied to an HPLCcolumn using a method reported above. The amount of material releasedfrom the conjugate was determined and expressed as percent of releasedmaterial.

In Vivo Efficacy

The therapeutic effect of the antibody-drug conjugate (ADC) compoundsand the improvement of their therapeutic efficacy in comparison with theparent drug, were assessed in animal models of human transplantedtumors. Mice bearing xenografts of human tumors were treated withsuitable doses of antibody-drug conjugates, of PNU-159682 free drug, andnaked antibody, at certain doses, and the tumor growth was recorded andcompared in the different treatment groups. FIGS. 30-32 show theefficacy of the antibody-drug conjugates of Formula Ic by xenografttumor inhibition in mice.

The efficacy of the antibody-drug conjugates of the invention may bemeasured in vivo by implanting allografts or xenografts of cancer cellsor primary tumors in rodents and treating the tumors with ADC accordingto the procedures of Example 12. Variable results are to be expecteddepending on the cell line, the specificity of antibody binding of theADC to receptors present on the cancer cells, dosing regimen, and otherfactors. For example, the in vivo efficacy of anti-HER2 ADC may bemeasured by a high expressing HER2 transgenic explant mouse model. Anallograft may be propagated from the Fo5 mmtv transgenic mouse whichdoes not respond to, or responds poorly to, HERCEPTIN® therapy. Subjectsare treated once with ADC and monitored over 3-6 weeks to measure thetime to tumor doubling, log cell kill, and tumor shrinkage. Follow updose-response and multi-dose experiments may be further conducted.

FIG. 30 shows a plot of the in vivo mean tumor volume change over timein Burkitt's lymphoma Bjab-luc xenograft tumors inoculated into CB17SCID mice after single dosing on day 0 with: (1) Vehicle, (2)thio-anti-CD22 (HC A114C)-MC-vc-PAB-MMAE 111 1 mg/kg, (3)thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102 1 mg/kg, (4)thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102 5 mg/kg, (5)thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107 1 mg/kg, (6)thio-anti-CD22 (HC A114C)-maleimide ketal-Ant 107 5 mg/kg, (7)thio-trastuzumab (HC A114C)-maleimide hydrazone-Ant 103 1 mg/kg, (8)thio-anti-CD22 (HC A114C)-maleimide hydrazone-Ant 108 1 mg/kg, (9)PNU-159682 free drug 8.77 ug/kg. All of the anti-CD22 conjugates (107,108 and 111) showed target-specific tumor growth inhibition and theinhibitory activity of the ketal-linked anti-CD22 ADC 107 wasdose-dependent. Free drug PNU-159682 and non-targeted control ADCs (102and 103) at the equivalent dose had no effect on tumor growth.

Table 5 shows the drug exposure level, average drug loading, tumorincidence, and responses for each test compound treated group of FIG. 30in the 48 day in vivo Bjab-luc xenograft tumor efficacy study. TheBjab-luc (EBV-negative Burkitt's lymphoma, luciferase expressing Bjabcells) tumors express CD22 receptor protein (Polson et al (2009) CancerRes. 69(6):2358-2364; US 2008/0050310; US 2005/0276812). CD22 isexpressed only in the B-cell compartment and on the surface of most NHLcells (D'Arena et al (2000) Am J Hematol. 64:275-81; Olejniczak et al(2006) Immunol Invest. 35:93-114). Efficacy in inhibiting the Bjab-lucxenograft tumor in mice as a model system may predict the clinicalresponse in treating patients with hematopoietic malignancies such asnon-Hodgkins lymphoma. All of the anti-CD22 ADC (107, 108, 111) wereeffective in tumor inhibition as compared with Vehicle and control ADC(102 and 103) that did not bind the Bjab-luc cells. The absence ofactivity with control ADC indicates that the activity seen with thetargeted ADC is specific, for example, it is not due to systemic releaseof free drug. In several cases, tumors were not only inhibited, but alsopartially and completely regressed by anti-CD22 conjugates (107, 108,111). These data indicate that anti-CD22 surface antigen is apotentially effective target for the anthracycline-derivative ADC of theinvention.

TABLE 5 in vivo Bjab-luc xenograft tumor efficacy study (FIG. 30) drugPR—partial CR—complete exposure avg. drug TI—tumor tumor tumor Testcompound dose ug/m2 loading incidence regression regression (1) Vehicle— — 9/9 0 0 (2) thio-anti-CD22 (HC 28.66 1.94 8/8 1 1 A114C)-MC-vc-PAB-MMAE 111 1 mg/kg (3) thio-trastuzumab (HC 26.65 2 9/9 0 0A114C)-maleimide ketal- Ant 102 1 mg/kg, (4) thio-trastuzumab (HC 133.252 9/9 0 0 A114C)-maleimide ketal- Ant 102 5 mg/kg (5) thio-anti-CD22 (HC26.42 2 9/9 0 0 A114C)-maleimide ketal- Ant 107 1 mg/kg (6)thio-anti-CD22 (HC 132.08 2 9/9 3 0 A114C)-maleimide ketal- Ant 107 5mg/kg (7) thio-trastuzumab (HC 22.65 1.7 9/9 0 0 A114C)-maleimidehydrazone-Ant 103 1 mg/kg (8) thio-anti-CD22 (HC 22.45 1.7 9/9 1 1A114C)-maleimide hydrazone-Ant 108 1 mg/kg (9) PNU-159682 free drug26.42 — 9/9 0 0 8.77 ug/kg

FIG. 31 shows a plot of the in vivo mean tumor volume change over timein MMTV-HER2Fo5 mammary allograft tumors inoculated into CRL nu/nu miceafter single dosing on day 0 with: (1) Vehicle, (2) trastuzumab-MCC-DM1101 5 mg/kg, (3) trastuzumab-MCC-DM1 101 10 mg/kg, (4) thio-trastuzumab(HC A114C)-maleimide ketal-Ant 102 5 mg/kg, (5) thio-trastuzumab (HCA114C)-maleimide ketal-Ant 102 10 mg/kg, (6) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 5 mg/kg, (7) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 10 mg/kg, (8) trastuzumab-MCC-DM1 101 5mg/kg+thio-trastuzumab (HC A114C)-maleimide ketal-Ant 102 5 mg/kg. Allof the anti-Her2 conjugates (101 and 102) showed target-specific tumorgrowth inhibition and the inhibitory activity was dose-dependent.Non-targeted control ADC 107 at equivalent doses had no effect on tumorgrowth.

Table 6 shows the tumor growth inhibition at day 7, drug exposure level,average drug loading, tumor incidence and responses for each testcompound treated group of FIG. 31 in the 38 day in vivo MMTV-HER2Fo5mammary allograft tumor efficacy study (Phillips et al (2008) CancerRes. 68(22):9280-9290; US 2005/0276812). The MMTV-HER2 Fo5 mammaryallograft tumor is a trastuzumab-insensitive, HER2-overexpressing breastcancer cell line. The targeted anti-HER2 ADC (101, 102, and combinationof 101 and 102) were effective in tumor inhibition as compared withVehicle and control ADC (107) that did not bind the MMTV-HER2 Fo5 cells.The absence of activity with control ADC indicates that the activityseen with the targeted ADC is specific, for example, it is not due tosystemic release of free drug. Tumors were not only inhibited, but alsopartially and completely regressed by anti-HER2 conjugates (101, 102,and combination of 101 and 102). Combination trastuzumab-MCC-DM1 101 andthio-trastuzumab (HC A114C)-maleimide-ketal-Ant 102 showed no additionalresponse beyond that of single agent 102. These data indicate thatanti-HER2 surface antigen is a potentially effective target for theanthracycline-derivative ADC of the invention.

TABLE 6 in vivo MMTV-HER2 Fo5 mammary allograft tumor efficacy study(FIG. 31) drug PR—partial CR—complete Test compound % inhibitionexposure avg. drug TI—tumor tumor tumor dose at day 7 ug/m2 loadingincidence regression regression (1) Vehicle — — — 8/8 0 0 (2)trastuzumab-MCC- 72 290 3.5 6/6 0 0 DM1 101 5 mg/kg (3) trastuzumab-MCC-86 580 3.5 6/6 0 0 DM1 101 10 mg/kg (4) thio-trastuzumab 87 110 1.5 6/73 2 (HC A114C)-maleimide ketal-Ant 102 5 mg/kg (5) thio-trastuzumab 91215 1.5 5/7 3 4 (HC A114C)-maleimide ketal-Ant 102 10 mg/kg (6)thio-anti-CD22 18 125 1.75  8/8 0 0 (HC A114C)-maleimide ketal-Ant 107 5mg/kg (7) thio-anti-CD22  5 250 1.75  8/8 0 0 (HC A114C)-maleimideketal-Ant 107 10 mg/kg (8) combination: 88 290 + 110 3.5 + 1.5 6/7 2 2trastuzumab-MCC- DM1, 101 5/mg/kg and thio-trastuzumab (HCA114C)-maleimide ketal-Ant 102, 5 mg/kg

FIG. 32 shows a plot of the in vivo mean tumor volume change over timein LnCap-Ner xenograft tumors inoculated into male SCID-beige mice aftersingle dosing on day 0 with: (1) Vehicle, (2) thio-anti-steap1 (HCA114C)-MC-vc-PAB-MMAE 112 1 mg/kg, (3) thio-anti-steap1 (HCA114C)-MC-vc-PAB-MMAE 112 3 mg/kg, (4) thio-anti-steap1 (HCA114C)-maleimide ketal-Ant 113 1 mg/kg, (5) thio-anti-steap1 (HCA114C)-maleimide ketal-Ant 113 3 mg/kg, (6) thio-anti-steap1 (HCA114C)-maleimide ketal-Ant 113 6 mg/kg, (7) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 1 mg/kg, (8) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 3 mg/kg, (9) thio-anti-CD22 (HCA114C)-maleimide ketal-Ant 107 6 mg/kg. All of the anti-steap1conjugates (112 and 113) showed target-specific tumor growth inhibitionand the inhibitory activity of the ketal-linked anti-steap1 ADC 113 wasdose-dependent. Non-targeted control ADC 107 at equivalent doses had noeffect on tumor growth.

Table 7 shows the drug exposure level, average drug loading, tumorincidence and responses for each test compound treated group of FIG. 32in the 49 day in vivo LnCap-Ner xenograft tumor efficacy study. Steap1(six-transmembrane epithelial antigen of the prostate) is aprostate-specific cell-surface antigen highly expressed in humanprostate tumors (Hubert et al (1999) PNAS 96(25):14523-14528). The LnCapcell line highly expresses steap1. The LnCap-Ner xenograft (Jin et al(2004) Cancer Res. 64:5489-5495) tumor inhibition study in mice may bean effective predictor for treatment of prostate cancer in patients. Thetargeted anti-steap1 ADC (112 and 113) were effective in tumorinhibition as compared with Vehicle and control ADC (107) that did notbind the LnCap cells. The absence of activity with control ADC indicatesthat the activity seen with the targeted ADC is specific, for example,it is not due to systemic release of free drug. Tumors were not onlyinhibited, but also partially regressed by anti-HER2 conjugates (112 and113). These data indicate that anti-steap1 surface antigen is apotentially effective target for the anthracycline-derivative ADC of theinvention.

TABLE 7 in vivo LnCap-Ner xenograft tumor efficacy study (FIG. 32) drugPR—partial CR—complete exposure avg. drug TI—tumor tumor tumor Testcompound dose ug/m2 loading incidence regression regression (1) Vehicle— — 8/8 0 0 (2) thio-anti-steap1 (HC 30.95 2 8/8 1 0 A114C)-MC-vc-PAB-MMAE 112 1 mg/kg (3) thio-anti-steap1 (HC 92.85 2 8/8 1 0A114C)-MC-vc- PAB-MMAE 112 3 mg/kg (4) thio-anti-steap1 (HC 22.83 1.658/8 0 0 A114C)-maleimide ketal- Ant 113 1/mg/kg (5) thio-anti-steap1 (HC68.49 1.65 8/8 1 0 A114C)-maleimide ketal- Ant 113 3 mg/kg (6)thio-anti-steap1 (HC 137 1.65 7/7 5 0 A114C)-maleimide ketal- Ant 113 6mg/kg (7) thio-anti-CD22 (HC 24.21 1.75 6/6 0 0 A114C)-maleimide ketal-Ant 107 1 mg/kg (8) thio-anti-CD22 (HC 72.64 1.75 7/7 1 0A114C)-maleimide ketal- Ant 107 3 mg/kg (9) thio-anti-CD22 (HC 145 1.757/7 0 0 A114C)-maleimide ketal- Ant 107 6 mg/kg

Rodent Toxicity

Antibody-drug conjugates and an ADC-minus control, “Vehicle”, may beevaluated in an acute toxicity rat model (Brown et al (2002) CancerChemother. Pharmacol. 50:333-340) and according to Example 11. Toxicityof ADC is investigated by treatment of female Sprague-Dawley rats withthe ADC and subsequent inspection and analysis of the effects on variousorgans. Based on gross observations (body weights), clinical pathologyparameters (serum chemistry and hematology) and histopathology, thetoxicity of ADC may be observed, characterized, and measured.

A multi-day acute toxicity study in adolescent female rats may beconducted by one or more doses of a candidate ADC, a control ADC, freeanthracycline derivative compound (PNU-159682) and a control Vehicle(day 0). Body weight is measured periodically. Clinical chemistry, serumenzymes and hematology analysis is also conducted periodically;concluding with complete necropsy with histopathological assessment.Toxicity signals included the clinical observation of weight loss,considering that weight loss, or weight change relative to animals dosedonly with Vehicle in animals after dosing with ADC, is a gross andgeneral indicator of systemic or localized toxicity. Hepatotoxicity maybe measured by: (i) elevated liver enzymes such as AST (aspartateaminotransferase), ALT (alanine aminotransferase), GGT (g-glutamyltransferase); (ii) increased numbers of mitotic and apoptotic figures;and (iii) hepatocyte necrosis. Hematolymphoid toxicity is observed bydepletion of leukocytes, primarily granuloctyes (neutrophils), and/orplatelets, and lymphoid organ involvement, i.e. atrophy or apoptoticactivity. Toxicity is also noted by gastrointestinal tract lesions suchas increased numbers of mitotic and apoptotic figures and degenerativeentercolitis.

Administration of Antibody-Drug Conjugate Pharmaceutical Formulations

Therapeutic antibody-drug conjugates (ADC) may be administered by anyroute appropriate to the condition to be treated. The ADC will typicallybe administered parenterally, i.e. infusion, subcutaneous,intramuscular, intravenous, intradermal, intrathecal, bolus, intratumorinjection or epidural (Shire et al (2004) J. Pharm. Sciences93(6):1390-1402). Pharmaceutical formulations of therapeuticantibody-drug conjugates (ADC) are typically prepared for parenteraladministration with a pharmaceutically acceptable parenteral vehicle andin a unit dosage injectable form. An antibody-drug conjugate (ADC)having the desired degree of purity is optionally mixed withpharmaceutically acceptable diluents, carriers, excipients orstabilizers, in the form of a lyophilized formulation or an aqueoussolution (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol,A. Ed.).

The ADC may be formulated as pharmaceutical compositions with apharmaceutically acceptable carrier or diluent. Any appropriate carrieror diluent may be used. Suitable carriers and diluents includephysiological saline solution and Ringers dextrose solutions.

Acceptable parenteral vehicles, diluents, carriers, excipients, andstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include: (i) buffers such as phosphate, citrate, dibasiccalcium phosphate, magnesium stearate, and other organic acids; (ii)antioxidants including ascorbic acid and methionine; (iii) preservatives(such as octadecyldimethylbenzyl ammonium chloride; hexamethoniumchloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; (iv) alkyl parabens such as methyl or propyl paraben;catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); (v) lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; (vi) hydrophilicpolymers such as polyvinylpyrrolidone; (vii) amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine; (viii)monosaccharides, disaccharides, and other carbohydrates includingglucose, lactose, sucrose, mannitol, trehalose, sodium starch glycolate,sorbitol mannose, carboxymethylcellulose, or dextrins; (ix) chelatingagents such as EDTA; (x) salt-forming counter-ions such as sodium; metalcomplexes (e.g. Zn-protein complexes); (xi) non-ionic surfactants suchas TWEEN™, PLURONICS™ or polyethylene glycol (PEG); (xii) glidants orgranulating agents such as magnesium stearate, carboxymethylcellulose,talc, silica, and hydrogenated vegetable oil; (xiii) disintegrant suchas crosprovidone, sodium starch glycolate or cornstarch; (xiv)thickening agents such as gelatin and polyethylene glycol; (xv) entericcoatings such as triethyl citrate; and/or (xvi) taste or texturemodifiers, antifoaming agents, pigments, and dessicants. For example,lyophilized anti-ErbB2 antibody formulations are described in WO97/04801, expressly incorporated herein by reference. An exemplaryformulation of an ADC contains about 100 mg/ml of trehalose(2-(hydroxymethyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-3,4,5-triol;C₁₂H₂₂O₁₁; CAS Number 99-20-7) and about 0.1% TWEEN™ 20 (polysorbate 20;dodecanoic acid2-[2-[3,4-bis(2-hydroxyethoxy)tetrahydrofuran-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethylester; C₂₆H₅₀O₁₀; CAS Number 9005-64-5) at approximately pH 6.

Pharmaceutical formulations of a therapeutic antibody-drug conjugate(ADC) may contain certain amounts of unreacted drug moiety (D),antibody-linker intermediate (Ab-L), and/or drug-linker intermediate(D-L), as a consequence of incomplete purification and separation ofexcess reagents, impurities, and by-products, in the process of makingthe ADC; or time/temperature hydrolysis or degradation upon storage ofthe bulk ADC or formulated ADC composition. For example, a formulationof the ADC may contain a detectable amount of free drug. Alternatively,or in addition to, it may contain a detectable amount of drug-linkerintermediate. Alternatively, or in addition to, it may contain adetectable amount of the antibody. The active pharmaceutical ingredientsmay also be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi permeable matrices of solidhydrophobic polymers containing the ADC, which matrices are in the formof shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(US 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT™ (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),and poly-D-(−)-3-hydroxybutyric acid.

Formulations may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy.Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared under sterile conditions and byuniformly and intimately bringing into association the ADC with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Aqueous suspensions contain the active materials (ADC) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

The pharmaceutical compositions of ADC may be in the form of a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, anaqueous solution intended for intravenous infusion may contain fromabout 3 to 500 μg of the active ingredient per milliliter of solution inorder that infusion of a suitable volume at a rate of about 30 mL/hr canoccur. Subcutaneous (bolus) administration may be effected with about1.5 ml or less of total volume and a concentration of about 100 mg ADCper ml. For ADC that require frequent and chronic administration, thesubcutaneous route may be employed, such as by pre-filled syringe orautoinjector device technology.

As a general proposition, the initial pharmaceutically effective amountof ADC administered per dose will be in the range of about 0.01-100mg/kg, namely about 0.1 to 20 mg/kg of patient body weight per day, withthe typical initial range of compound used being 0.3 to 15 mg/kg/day.For example, human patients may be initially dosed at about 1.5 mg ADCper kg patient body weight. The dose may be escalated to the maximallytolerated dose (MTD). The dosing schedule may be about every 3 weeks,but according to diagnosed condition or response, the schedule may bemore or less frequent. The dose may be further adjusted during thecourse of treatment to be at or below MTD which can be safelyadministered for multiple cycles, such as about 4 or more.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Although oral administration of protein therapeutics are generallydisfavored due to poor bioavailability due to limited absorption,hydrolysis or denaturation in the gut, formulations of ADC suitable fororal administration may be prepared as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the ADC.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Exemplary unit dosage formulations contain a dailydose or unit daily sub-dose, or an appropriate fraction thereof, of theactive ingredient.

Antibody-Drug Conjugate Methods of Treatment

Antibody-drug conjugates of the invention are useful as antitumoragents. A mammal, e.g. a human or animal, may therefore be treated by amethod comprising administering thereto a pharmaceutically effectiveamount of a conjugate of formula I as hereinbefore defined. Thecondition of the human or animal may be ameliorated or improved in thisway.

Formula I ADC may be used to treat various diseases or disorders in apatient, such as cancer and autoimmune conditions including thosecharacterized by the overexpression of a tumor-associated antigen.Exemplary conditions or disorders include benign or malignant tumors;leukemia and lymphoid malignancies; other disorders such as neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologicdisorders. Cancer types susceptible to ADC treatment include those whichare characterized by the overexpression of certain tumor associatedantigens or cell surface receptors, e.g. HER2.

One method is for the treatment of cancer in a mammal, wherein thecancer is characterized by the overexpression of an ErbB receptor. Themammal optionally does not respond, or responds poorly, to treatmentwith an unconjugated anti-ErbB antibody. The method comprisesadministering to the mammal a therapeutically effective amount of anantibody-drug conjugate compound. The growth of tumor cells thatoverexpress a growth factor receptor such as HER2 receptor or EGFreceptor may be inhibited by administering to a patient a Formula I ADCwhich binds specifically to said growth factor receptor and achemotherapeutic agent wherein said antibody-drug conjugate and saidchemotherapeutic agent are each administered in amounts effective toinhibit growth of tumor cells in the patient.

A human patient susceptible to or diagnosed with a disordercharacterized by overexpression of ErbB2 receptor, may be treated byadministering a combination of a Formula I ADC and a chemotherapeuticagent. Such excessive activation may be attributable to overexpressionor increased production of the ErbB receptor or an ErbB ligand. In oneembodiment, a diagnostic or prognostic assay will be performed todetermine whether the patient's cancer is characterized by excessiveactivation of an ErbB receptor. For example, ErbB gene amplificationand/or overexpression of an ErbB receptor in the cancer may bedetermined. Various assays for determining suchamplification/overexpression are available in the art and include IHC,FISH and shed antigen assays.

Examples of cancer to be treated herein include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, gastrointestinal stromal tumor(GIST), pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The ADC formulation is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of ADC is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dosageregimen might range from about 1 μg/kg to 100 mg/kg or more, dependingon the factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of disease symptoms occurs. An exemplary dosingregimen comprises administering an initial loading dose of about 4mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of theADC. Other dosage regimens may be useful.

Combination Therapy

An antibody-drug conjugate (ADC) may be combined in a pharmaceuticalcombination formulation, or dosing regimen as combination therapy, witha second compound having anti-cancer properties. The second compound ofthe pharmaceutical combination formulation or dosing regimen preferablyhas complementary activities to the ADC of the combination such thatthey do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent,cytokine, growth inhibitory agent, anti-hormonal agent, aromataseinhibitor, protein kinase inhibitor, lipid kinase inhibitor,anti-androgen, antisense oligonucleotide, ribozyme, gene therapyvaccine, anti-angiogenic agent and/or cardioprotectant. Such moleculesare suitably present in combination in amounts that are effective forthe purpose intended. A pharmaceutical composition containing an ADC mayalso have a therapeutically effective amount of a chemotherapeutic agentsuch as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNAbinder.

Alternatively, or additionally, the second compound may be an antibodywhich binds or blocks ligand activation of tumor-associated antigen orreceptor. The second antibody may be conjugated with a cytotoxic orchemotherapeutic agent, e.g., a macrocyclic depsipeptide, an auristatin,a calicheamicin, or a 1,8 bis-naphthalimide moiety. For example, it maybe desirable to further provide antibodies which bind to EGFR, ErbB2,ErbB3, ErbB4, or vascular endothelial factor (VEGF) in the oneformulation or dosing regimen.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein there is a time period while both (or all) activeagents simultaneously exert their biological activities.

In one embodiment, treatment with an ADC of the present inventioninvolves the combined administration of an anticancer agent identifiedherein, and one or more chemotherapeutic agents or growth inhibitoryagents. Preparation and dosing schedules for such chemotherapeuticagents may be used according to manufacturers's instructions or asdetermined empirically by the skilled practitioner. Preparation anddosing schedules for such chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992).

The ADC may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (EP 616812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated ishormone independent cancer, the patient may previously have beensubjected to anti-hormonal therapy and, after the cancer becomes hormoneindependent, the anti-ErbB2 antibody (and optionally other agents asdescribed herein) may be administered to the patient. It may bebeneficial to also coadminister a cardioprotectant (to prevent or reducemyocardial dysfunction associated with the therapy) or one or morecytokines to the patient. In addition to the above therapeutic regimes,the patient may be subjected to surgical removal of cancer cells and/orradiation therapy.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Metabolites of the Antibody-Drug Conjugates

Also falling within the scope of this invention are the in vivometabolic products of the ADC compounds described herein, to the extentsuch products are novel and unobvious over the prior art. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification, enzymatic cleavage, and the like, of theadministered compound. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products may be identified by preparing a radiolabelled (e.g.¹⁴C or ³H) ADC, administering it parenterally in a detectable dose (e.g.greater than about 0.5 mg/kg) to an animal such as rat, mouse, guineapig, monkey, or to man, allowing sufficient time for metabolism to occur(typically about 30 seconds to 30 hours) and isolating its conversionproducts from the urine, blood or other biological samples. Theseproducts are easily isolated since they are labeled (others are isolatedby the use of antibodies capable of binding epitopes surviving in themetabolite). The metabolite structures are determined in conventionalfashion, e.g. by MS, LC/MS or NMR analysis. In general, analysis ofmetabolites is done in the same way as conventional drug metabolismstudies well-known to those skilled in the art. The conversion products,so long as they are not otherwise found in vivo, are useful indiagnostic assays for therapeutic dosing of the ADC compounds.

Metabolites include the products of in vivo cleavage of the ADC wherecleavage of any bond occurs that links the drug moiety to the antibody.Metabolic cleavage may thus result in the naked antibody, or an antibodyfragment. The antibody metabolite may be linked to a part, or all, ofthe linker. Metabolic cleavage may also result in the production a drugmoiety or part thereof. The drug moiety metabolite may be linked to apart, or all, of the linker.

Articles of Manufacture

In another embodiment, an article of manufacture, or “kit”, containingADC and materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, or blisterpack. The containers may be formed from a variety of materials such asglass or plastic. The container holds an antibody-drug conjugate (ADC)composition which is effective for treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anADC. The label or package insert indicates that the composition is usedfor treating the condition of choice, such as cancer.

In one embodiment, the article of manufacture may further comprise asecond (or third) container comprising a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution, anda package insert indicating that the first and second compounds can beused to treat cancer. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

EXAMPLES

The compounds of the present invention, as prepared according to thefollowing examples, were characterized by HPLC/MS analytical data;HPLC/MS data were collected following any one of methods 1, 2.

HPLC/MS Analytic Method 1

Waters 2795 Alliance HT HPLC system equipped with a 2996 Waters PDAdetector and Micromass mod. ZQ single quadruple mass spectrometer,equipped with an electrospray (ESI) ion source. Instrument control, dataacquisition and data processing were provided by Empower and MassLynx4.0 software. HPLC was carried out at 30° C. at a flow rate of 1.0mL/min using a C18, 3 micron Phenomenex (4.6×50 mm) column. Mobile phaseA was ammonium acetate 5 mM pH 5.2 buffer with acetonitrile (95:5), andmobile phase B was H₂O/acetonitrile (5:95); the gradient was from 10 to90% B in 8 minutes then ramp to 100% B in 1.0 minutes. The injectionvolume was 10 uL. The mass spectrometer was operated in positive and innegative ion mode, the capillary voltage was set up at 3.5 KV (ES⁺) and28 V (ES); the source temperature was 120° C.; cone was 14 V (ES⁺) and2.8 KV (ES); full scan, mass range from 100 to 1000 m/z.

HPLC/MS Analytic Method 2

Waters 2795 HPLC system was equipped with a 996 Waters PDA detector andMicromass mod. ZQ single quadruple mass spectrometer, equipped with anelectrospray (ESI) ion source. Instrument control, data acquisition anddata processing were provided by Empower and MassLynx 4.0 software. HPLCwas carried out at 30° C. at a flow rate of 1 mL/min using a RP18 WatersX Terra (4.6 μM×50 mm) column. Mobile phase A was ammonium hydroxide0.05% pH=10 buffer with acetonitrile (95:5), and Mobile phase B wasH₂O/acetonitrile (5:95); the gradient was from 10 to 90% B in 8 minutesthen hold 100% B for 2 minutes. The injection volume was 10 4. The massspectrometer was operated in positive and in negative ion mode, thecapillary voltage was set up at 2.5 KV; the source temperature was 120°C.; cone was 10 V; full scan, mass range from 100 to 1000 m/z.

Example 1N-methyl-2-[(1-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetamide(Compound 2) Step 1 Synthesis of the intermediate ethyl[(1-{2-oxo-2-[(2S,45)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetate45

To a solution of(8S,10S)-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-7,8,9,10-tetrahydrotetracene-5,12-dione(50 mg, 0.078 mmol) [PNU-159682, compound IIA, prepared as reported inWO 9802446] in 2 ml of dry dimethylformamide kept under argon,(Cyclohex-1-enyloxy)-acetic acid ethyl ester (0.5 mL, [prepared asreported in J. Org. Chem. (1978) 43:1244-1245] and p-toluenesulfonicacid monohydrate (5 mg, 0.026 mmol) were added. The reaction mixture wasstirred overnight at room temperature, sodium bicarbonate saturatedsolution was added (20 mL) and the product extracted withdichloromethane (2×20 mL). The combined organic phases were dried overanhydrous sodium sulfate, filtered, the solvent removed under vacuum andthe residue partially purified by flash chromatography (DCM/MeOH97.5:2.5) to give 30 mg (37%) of the ester intermediate that was used assuch in step 2. MS (ESI): 826 [M+H]⁺. Retention time=7.48 min. method 2

Step 2 Synthesis of the intermediate[(1-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]aceticacid 46

To 30 mg of 45, 5 mL of in 01N NaOH was added. The suspension was cooledat 5° C. and stirred under argon for 3 hours. The aqueous solution wasbrought to pH≅8 with 10% acetic acid water solution and extracted withdichloromethane (2×10 mL). The combined organic phases were dried overanhydrous sodium sulfate, filtered, the solvent removed under vacuum andthe residue purified by flash chromatography (DCM/MeOH 90:10) to give 5mg (y=8% 2 steps) of the acid intermediate 46 as a red solid. MS (ESI):798 [M+H]⁺. Retention time=3.94 min. Method 2

Step 3 Synthesis of intermediate1-({[(1-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetyl}oxy)pyrrolidine-2,5-dione47

To a solution of the acid intermediate 46 (4 mg, 0.005 mmol) in dryDichloromethane (2 mL) kept at +5° C., N-hydroxysuccinimide (2 mg, 0.017mmol) and N,N′-dicyclohexylcarbodiimide (2 mg, 0.01 mmol) were added.The solution was stirred at room temperature for 6 h, the solventevaporated under vacuum and the residue treated with ethyl ether (5 mL).The suspension was stirred for 30 minutes, the solid removed byfiltration and organic solution concentrated in vacuo. Purification ofthe crude by flash chromatography (DCM/Acetone 80:20) yield 1.5 mg(y=33%) of 47 as a red solid. MS (ESI): 895 [M+H]⁺. Retention time=3.22min. Method 1.

Step 4 Synthesis of the Title Compound 2

To a solution of 47 obtained from step 3 (1 mg, 0.001 mmol) in drytetrahydrofuran (2 mL), 1 M methylamine in THF (3 μL, 0.003 mmol) wereadded. The solution was stirred at room temperature 30 minutes, thesolvent evaporated under vacuum and the residue purified by flashchromatography (dichloromethane/methanol 90:10) yield 0.9 mg (y=99%) of2 as a red solid. MS (ESI): 811 [M+H] Retention time=6.10 min. Method 2,Retention time=5.99 min. Method 1.

By analogous procedure and using the suitable starting materials thefollowing compounds were prepared:

2-[(1-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetamide(Compound 1) MS (ESI): 887 [M+H] Retention time=5.86 min. Method 2

N-benzyl-2-[(1-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetamide(Compound 3) MS (ESI): 797 [M+H] Retention time=7.16 min. Method 2

N²-(tert-butoxycarbonyl)-N⁶-{[(1-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetyl}-L-lysine(Compound 4) MS (ESI): 1026 [M+H]⁺. Retention time=5.26 min. Method 1;Retention time=4.64 min. Method 2

Example 2N-methyl-2-[(6-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}tetrahydro-2H-pyran-2-yl)methoxy]acetamide(Compound 7) Step 1 Synthesis of the intermediate: ethyl(3,4-dihydro-2H-pyran-2-ylmethoxy)acetate

In a dried round bottomed flask under argon atmosphere, 60% sodiumhydride (240 mg, 6.0 mmol) was rinsed three times with anhydrousn-pentane. A solution of 2-hydroxymethyl-3,4-dihydro-2H-pyran (570.8 mg,5 mmol) in tetrahydrofuran (10 ml) was cooled at 0° C. and then added tothe NaH. The reaction mixture was stirred at 0° C. until hydrogenevolution ended. A solution of ethyl bromoacetate (1253 mg, 7.5 mmol) intetrahydrofuran (6 ml) was added to the reaction mixture and thestirring was continued at room temperature until disappearance of thestarting alcohol (TLC analysis). After cooling, H₂O was added, and thesolvent was evaporated under reduced pressure. The residue was purifiedby flash column chromatography (AcOEt:hexane=1:12) on silica gel(230-400 mesh), affording 626 mg (yield 57%) of ethyl(3,4-dihydro-2H-pyran-2-ylmethoxy)acetate as a colorless oil; ¹H NMR(401 MHz, DMSO-d₆) δ ppm 1.18-1.23 (m, 3H) 3.55-3.59 (m, 2 H) 3.93 (m,J=10.08, 5.11, 5.11, 2.38 Hz, 1H) 4.13 (q, J=7.07 Hz, 2H) 4.14 (s, 2H)4.67 (dddd, J=6.14, 4.83, 2.56, 1.34 Hz, 1H) 6.36 (dt, J=6.13, 1.75 Hz,1H).

Step 2 Synthesis of the intermediate: ethyl[(6-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}tetrahydro-2H-pyran-2-yl)methoxy]acetate48

To a solution of(8S,10S)-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-7,8,9,10-tetrahydrotetracene-5,12-dione(50 mg, 0.078 mmol) [PNU-159682, formula IIA, prepared as reported in WO9802446] in 4 ml of dry dichloromethane under argon atmosphere, ethyl(3,4-dihydro-2H-pyran-2-ylmethoxy)acetate from step 1 (118.5 mg, 0.592mmol) and anhydrous p-toluenesulfonic acid (22.3 mg, 0.12 mmol) wereadded. The reaction mixture was stirred at room temperature for 4 hours,until no starting material was detectable (TLC analysis,MeOH:CH₂Cl₂=0.3:9.7). Sodium bicarbonate 10% aqueous solution was thenadded to the reaction mixture and the aqueous phase was extracted withdichloromethane (4×20 ml).The combined organic phases were dried overanhydrous sodium sulfate, filtered and evaporated under vacuum. Theresidue was purified by flash column chromatography(MeOH:CH₂Cl₂=0.3:9.7) on silica gel (230-400 mesh), affording 41 mg (redwax, yield 62%) of 48, as a mixture of four diastereoisomers. MS (ESI):842 [M+H]⁺. Retention time=7.47, 7.83 min (method 2).

Step 3 Synthesis of the intermediate:[(6-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}tetrahydro-2H-pyran-2-yl)methoxy]aceticacid 49

The ethyl ester intermediate 48 obtained from step 2 (40 mg, 0.0475mmol) cooled at 0° C., was treated with aqueous 0.1 N sodium hydroxide(1.5 ml) under argon. The reaction mixture was stirred at 0° C. for 2hours. The course of the reaction was followed by reverse-phase HPLC-MS.After that, the reaction mixture was brought to pH≅ 8 with 10% aceticacid water solution, and extracted with n-butanol saturated with water(8×10 mL). The combined organic phases were dried over anhydrous sodiumsulfate, filtered, and the solvent removed under vacuum. The residue waspurified by flash column chromatography (MeOH:CH₂Cl₂=1:9) on silica gel(230-400 mesh), affording 4.5 mg (red solid, yield 12%) of 49 as adiasteroisomeric mixture. MS (ESI): 814 [M+H]⁺. Retention time=2.99,3.50, 3.66 (method 2). Retention time=4.24 (method 1).

Step 4 Synthesis of the intermediate:1-({[(6-{2-oxo-2-[(2S,45)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}tetrahydro-2H-pyran-2-yl)methoxy]acetyl}oxy)pyrrolidine-2,5-dione50

To a solution of the acid intermediate 49 obtained from step 3 of theprocess (2 mg, 0.0024 mmol) in dry dichloromethane (1 ml) cooled at 0°C., N-hydroxysuccinimide (1 mg, 0.00792 mmol) andN,N′-dicyclohexylcarbodiimide (1 mg, 0.004556 mmol) were added. Thereaction mixture was stirred at 0° C. for 2.5 h, until disappearance ofthe starting material (HPLC-MS analysis). The solvent evaporated undervacuum and the residue treated with ethyl ether (2×4 ml). The suspensionwas stirred for 10 minutes, the solid removed by filtration and theorganic solution concentrated in vacuo, affording 2 mg of 50 (red solid)as a mixture of diastereoisomers. MS (ESI): 911 [M+H]⁺. Retentiontime=6.12, 6.30, 6.42 min (method 1).

Step 5 The Title Compound 7

To a solution of 50 obtained from step 4 of the process (1 mg, 0.0011mmol) in dry dichloromethane (100 μL), 2.0 M methylamine in THF (65 μL,0.0033 mmol) were added. The solution was stirred at room temperature 4hours until no starting material was detectable (HPLC-MS analysis), andthe solvent evaporated under vacuum. The residue was purified by flashcolumn chromatography (MeOH: CH₂Cl₂=0.3:9.7) on silica gel (230-400mesh), affording 0.46 mg (red solid, yield 51%) of 7 as a mixture ofdiastereoisomers. MS (ESI): 827 [M+H]⁺. Retention time=5.34, 5.54, 5.66min (method 1).

By analogous procedures and using the suitable starting materials thefollowing compounds were prepared:

2-[(6-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}tetrahydro-2H-pyran-2-yl)methoxy]acetamide(Compound 6) MS (ESI): 813 [M+H]⁺. Retention time=5.42 min (method 2).

N-benzyl-2-[(6-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}tetrahydro-2H-pyran-2-yl)methoxy]acetamide(Compound 8) MS (ESI): 903 [M+H]⁺. Retention time=6.92 min (method 1).Retention time=6.94 min (method 2).

Example 3N-acetyl-3-({3-[(2E)-2-{2-hydroxy-1-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethylidene}hydrazinyl]-3-oxopropyl}disulfanyl)-L-alanine(Compound 12) Step 1N′-{(1E)-2-hydroxy-1-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethylidene}-3-(pyridin-2-yldisulfanyl)propanehydrazide53

A solution of 3-(2-pyridyldithio)propionic acid hydrazide HCl (41.5 mg,0.156 mmol) in anhydrous methanol (5 ml) was added to(8S,10S)-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-7,8,9,10-tetrahydrotetracene-5,12-dione[PNU-159682, compound IIA, prepared as reported in WO 9802446] (50 mg,0.078 mmol). The solution was stirred in the dark at room temperaturefor 20 hours. The course of the reaction was followed by reverse-phaseHPLC-MS. After this period the solvent was evaporated and the residuewas purified by flash column chromatography (MeOH:CH₂Cl₂=0.2:9.8) onsilica gel (230-400 mesh), affording 18 mg (yield 27%) of 53. MS (ESI):853 [M+H]⁺. Retention time=5.52 min (method 2).

By analogous procedures and using the suitable starting materials thefollowing compound was prepared:6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-{(1E)-2-hydroxy-1-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethylidene}hexanehydrazide52. MS (ESI): 849 [M+H]⁺. Retention time=5.15 min (method 2).

Step 2

To a solution of 53 obtained from step 1 (8.5 mg, 0.01 mmol.)N-acetylcysteine was added (0.32 mg, 0.02 mmol). The solution wasstirred at room temperature for 24 hours, the solvent was evaporated andthe residue was purified by flash column chromatography(MeOH:CH₂Cl₂=2:8) on silica gel (230-400 mesh), affording 7.2 mg (yield80%) of 12 as a red solid. MS (ESI): 905 [M+H]⁺. Retention time=3.62 min(method 2).

By analogous procedures and using the suitable starting materials thefollowing compounds were prepared:

N-acetyl-S-(1-{6-[(2E)-2-{2-hydroxy-1-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethylidene}hydrazinyl]-6-oxohexyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine(Compound 9). MS (ESI): 1012 [M+H]⁺.

N²-(tert-butoxycarbonyl)-N⁶-(1-{6-[(2E)-2-{2-hydroxy-1-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethylidene}hydrazinyl]-6-oxohexyl}-2,5-dioxopyrrolidin-3-yl)-L-lysine(Compound 10, Table 1) MS (ESI): 1095 [M+H]⁺.

Example 3a Preparation of(2S,45)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide54

To a solution of PNU-159682 (15.3 mg, 0.02038 mmol) prepared as reportedin WO 98/02446, in 3 ml of methanol and 2 ml of H₂O, a solution of NaIO₄(5.1 mg, 0.0238 mmol) in 1 ml of H₂O was added. The reaction mixture wasstirred at room temperature for 3 hours, until no starting material wasdetectable (TLC and HPLC analysis). The solvents were removed underreduced pressure and the crude red solid(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxylicacid 56 was used without further purifications in the next step. MS(ESI): 628 [M+H]⁺. Retention time=2.1-3.2 min (method 1, Example 3b).

To a solution of the crude intermediate 56 (4.4 mg) in anhydrousdichloromethane (1.5 ml) under argon atmosphere, anhydrous triethylamine(2.2 mg, 0.0204 mmol), TBTU (4.4 mg, 0.01388 mmol) and commerciallyavailable N-(2-aminoethyl)maleimide trifluoroacetate salt (3.6 mg,0.00694 mmol) were added. The reaction mixture was stirred at roomtemperature for 30 min, until disappearance of the starting material(HPLC-MS analysis). The solvent was evaporated under vacuum and theresidue was then purified by flash column chromatography(EtOH:CH₂Cl₂=0.2:9.8) on silica gel (230-400 mesh), affording 1.1 mg(red solid, yield calculated on PNU-159682=21%) of 54. MS (ESI): 750[M+H]⁺. Retention time=5.18 min (method 1, Example 3b).

Example 3b Preparation ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-{[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]carbonyl}piperazin-1-yl)carbonyl]oxy}methyl)phenyl]-L-ornithinamide55

Step 14-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl4-nitrophenyl carbonate 58 (30 mg, 0.041 mmol) was reacted withtert-butyl piperazine-1-carboxylate (5.3 mg, 0.0287 mmol) in anhydrousDMSO under argon atmosphere at room temperature (FIG. 7 d). The reactionmixture was stirred for 1 h, until disappearance of the startingmaterial (HPLC-MS analysis). Diethyl ether (80 ml) was then added to thereaction mixture and the precipitate thus obtained was collected byfiltration to giveN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[({[4-(tert-butoxycarbonyl)piperazin-1-yl]carbonyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide59 as a yellow solid, 22.0 mg) was isolated and used without furtherpurification in the next step. MS (ESI): 785 [M+H]⁺. Retention time=4.87min (method 1).

Step 2 The intermediate 59 (22.0 mg) was treated with trifluoroaceticacid (327 mg, 2.87 mmol) in anhydrous dichloromethane (0.12 ml). Thereaction mixture was stirred at room temperature for 15 minutes, untildisappearance of the starting material (HPLC-MS analysis). After that,the reaction mixture was treated with diethyl ether (20 ml) and theresidue thus obtained was rinsed with diethyl ether (2×10 ml): theproductN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-(4-{[(piperazin-1-ylcarbonyl)oxy]methyl}phenyl)-L-ornithinamide60 (white wax, 20.0 mg) was thus isolated and used without furtherpurification in the next step. MS (ESI): 685 [M+H]⁺. Retention time=2.97min (method 1).

Step 3 To the intermediate 60 (13.2 mg) a solution of crude(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxylicacid 56 (9.3 mg) in anhydrous dichloromethane (2.6 ml), TBTU (5.3 mg,0.0165 mmol), and anhydrous triethylamine (2.8 mg, 0.0275 mg) was added.The reaction mixture was stirred at room temperature under argonatmosphere for 15 minutes, until disappearance of the starting material(HPLC-MS analysis). The solvent was then evaporated under vacuum and thecrude was purified by flash column chromatography (EtOH:AcOEt=1.5:8.5)on silica gel (230-400 mesh), affording 4.8 mg (red solid, yieldcalculated on PNU-159682=34%) of 55. MS (ESI): 1295 [M+H]⁺. Retentiontime=5.51 min(method 1).

Compounds were characterized by HPLC/MS analytical data; HPLC/MS datawere collected following any one of the following Methods 1 or 2.

HPLC/MS Analytic Method 1: The HPLC equipment consisted of a Waters 2795Alliance HT system equipped with a 2996 Waters PDA detector andMicromass mod. ZQ single quadrupole mass spectrometer, equipped with anelectrospray (ESI) ion source. Instrument control, data acquisition anddata processing were providen by Empower and MassLynx 4.0 software. HPLCwas carried out at 30° C. at a flow rate of 1.0 mL/min using a Waters XTerra MS C18-3.5 nM (4.6×50 mm) column. Mobile phase A was ammoniumacetate 5 mM pH=5.2 buffer with acetonitrile (95:5), and mobile phase Bwas H₂O/acetonitrile (5:95); the gradient was from 10 to 90% B in 8minutes then ramp to 100% B in 1.0 minutes. The mass spectrometer wasoperated in positive and in negative ion mode, the capillary voltage wasset up at 3.5 kV (ES⁺) and 28 V (ES⁻); the source temperature was 120°C.; cone was 14 V (ES⁺) and 2.8 kV (ES⁻); full scan, mass range from 100to 1000 m/z was set up.

HPLC/MS Analytic Method 2: The HPLC equipment consisted of a Waters 2795HPLC system equipped with a 996 Waters PDA detector and Micromass mod.ZQ single quadrupole mass spectrometer, equipped with an electrospray(ESI) ion source. Instrument control, data acquisition and dataprocessing were provided by Empower and MassLynx 4.0 software. HPLC wascarried out at 30° C. at a flow rate of 1 mL/min using a RP18 Waters XTerra (4.6 μM×50 mm) column. Mobile phase A was ammonium hydroxide 0.05%pH=10 buffer with acetonitrile (95:5), and Mobile phase B wasH₂O/acetonitrile (5:95); the gradient was from 10 to 90% B in 8 minutesthen hold 100% B for 2 minutes. The mass spectrometer was operated inpositive and in negative ion mode, the capillary voltage was set up at2.5 kV; the source temperature was 120° C.; cone was 10 V; full scan,mass range from 100 to 1000 m/z was set up.

Example 3c Preparation of(8S,10S)-6,8,11-trihydroxy-1-methoxy-10-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-8-(piperazin-1-ylcarbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione57

To a solution of 56 (9 mg) in anhydrous dichloromethane (5 ml) underargon atmosphere, anhydrous triethylamine (1.6 mg, 0.0158 mmol),piperazine (3.6 mg, 0.0424 mmol), HOBt (2.1 mg, 0.0158 mmol) and EDC(3.0 mg, 0.0158 mmol) were added. The reaction mixture was stirred atroom temperature over night. The solvent was evaporated under vacuum andthe residue was then purified by flash column chromatography(DCM/MeOH/AcOH/H₂O 45/4/1/0.5) on silica gel (230-400 mesh). The productobtained was dissolved in DCM and washed with satd. NaHCO₃ (x2) andwater (x2). The organic solvent was evaporated under vacuum to afford5.0 mg of 57. MS (ESI): 696 [M+H]⁺. Retention time=4.01 min (method 1,Example 3b).

Example 3d Preparation ofN-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-2-[(6-{2-oxo-2-[(2S,45)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6-methylidene-11-oxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}tetrahydro-2H-pyran-2-yl)methoxy]acetamide51

To a solution of the crude intermediate 50 (103.8 mg) in anhydrousdichloromethane (29.7 ml) under argon atmosphere, the commerciallyavailable N-(2-aminoethyl)maleimide trifluoroacetate salt (57.9 mg,0.228 mmol) and anhydrous triethylamine (23.1 mg, 0.228 mmol) wereadded. The reaction mixture was stirred at room temperature for 1 h,until disappearance of the starting material (HPLC-MS analysis). Thesolvent was evaporated under vacuum and the residue rinsed with amixture of Et₂O/n-hexane (1 ml/20 ml). The crude was then purified byflash column chromatography (EtOH:CH₂Cl₂=0.2:9.8) on silica gel (230-400mesh), affording 12.2 mg (red solid, yield calculated on PNU-159682=20%)of 51 as a diasteroisomeric mixture. MS (ESI): 936 [M+H]⁺. Retentiontime=5.86, according to the following method:

Waters 2795 Alliance HT HPLC system with a 2996 Waters PDA detector andMicromass mod. ZQ single quadrupole mass spectrometer, with anelectrospray (ESI) ion source. Instrument control, data acquisition anddata processing by Empower and MassLynx 4.0 software. HPLC was carriedout at 30° C. at a flow rate of 1.0 mL/min using a Waters X Terra MSC18-3.5 μM (4.6×50 mm) column. Mobile phase A was ammonium acetate 5 mMpH=5.2 buffer with acetonitrile (95:5), and mobile phase B wasH₂O/acetonitrile (5:95); the gradient was from 10 to 90% B in 8 minutesthen ramp to 100% B in 1.0 minutes. The mass spectrometer was operatedin positive and in negative ion mode, the capillary voltage was set upat 3.5 kV (ES⁺) and 28 V (ES); the source temperature was 120° C.; conewas 14 V (ES⁺) and 2.8 kV (ES); full scan, mass range from 100 to 1000m/z.

Example 4 Preparation of the MCM2 conjugate Compound 5, as identified inTable 1

MCM2 (10-294) protein [Ishimi et al (2001) Jour. Biol. Chem.276(46):42744-42752] (1.5 mg, 0.045 mmol) was dissolved in 0.5 ml ofphosphate buffered saline solution (pH 7.2), pH value was adjusted to8.5 by addition of 55 μl of 1M NaHCO₃ (pH 8.5) and 0.5 mg of1-({[(1-{2-oxo-2-[(2S,45)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetyl}oxy)pyrrolidine-2,5-dione(0.55 μmol) [prepared as reported in example 1 step 3] was added from a10 mg/ml acetonitrile solution. The reaction was incubated for 1 hr atroom temperature then the reaction mixture was desalted on a NAP-10column conditioned in phosphate buffered saline solution and thefractions containing the protein were collected and pooled.

The reacted protein was analyzed by SDS PAGE in comparison withunreacted MCM2 and different amount of1-({[(1-{2-oxo-2-[(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl]ethoxy}cyclohexyl)oxy]acetyl}oxy)pyrrolidine-2,5-dioneas reported above.

By analogous procedures and using the suitable starting materials thefollowing compounds (Table 1) were prepared: MCM2 conjugate Compound 11;MCM2 conjugate Compound 13; MCM2 conjugate Compound 14.

Example 5 Stability of the Conjugate: General Procedure

To 0.5 mg of conjugate, ammonium acetate 5 mM pH=5.2 buffer solution(200 mL) was added. The solution was warmed at 37° C. and sample wastaken periodically and analyzed by HPLC (method 2). Results areexpressed as % of released material(8S,10S)-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-7,8,9,10-tetrahydrotetracene-5,12-dione(Compound IIA) from the conjugate.

By analogous procedures, using the ammonium acetate 5 mM pH=4.5 buffersolution, the stability at pH 4.5 was also determined.

By analogous procedures, stability was performed on Compound 12 (Table1), showing, after four hours incubation, 90% release in pH 5.2 buffersolution and 100% release in pH 4.2 buffer solution, of(8S,10S)-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-10-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-7,8,9,10-tetrahydrotetracene-5,12-dione(Compound IIA) from the conjugate.

Example 6 Preparation of Cysteine Engineered Antibodies for Conjugationby Reduction and Reoxidation

Light chain and heavy chain amino acids are numbered according to Kabat(Kabat et al., Sequences of proteins of immunological interest, (1991)5th Ed., US Dept of Health and Human Service, National Institutes ofHealth, Bethesda, Md.). Single letter amino acid abbreviations are used.

Full length, cysteine engineered monoclonal antibodies (ThioMabs)expressed in CHO cells bear cysteine adducts (cystines) orglutathionylated on the engineered cysteines due to cell cultureconditions. To liberate the reactive thiol groups of the engineeredcysteines, the ThioMabs are dissolved in 500 mM sodium borate and 500 mMsodium chloride at about pH 8.0 and reduced with about a 50-100 foldexcess of 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getzet al (1999) Anal. Biochem. 273:73-80; Soltec Ventures, Beverly, Mass.)for about 1-2 hrs at 37° C. Alternatively, DTT can be used as reducingagent. The formation of interchain disulfide bonds was monitored eitherby non-reducing SDS-PAGE or by denaturing reverse phase HPLC PLRP columnchromatography. The reduced cysteine engineered antibody is diluted andloaded onto a HiTrap S column in 10 mM sodium acetate, pH 5, and elutedwith PBS containing 0.3M sodium chloride. The eluted reduced cysteineengineered antibody (ThioMab) is treated with 2 mM dehydroascorbic acid(dhAA) at pH 7 for 3 hours, or 2 mM aqueous copper sulfate (CuSO₄) atroom temperature overnight. Ambient air oxidation may also be effective.The buffer is exchanged by elution over Sephadex G25 resin and elutedwith PBS with 1 mM DTPA. The thiol/Ab value is checked by determiningthe reduced antibody concentration from the absorbance at 280 nm of thesolution and the thiol concentration by reaction with DTNB (Aldrich,Milwaukee, Wis.) and determination of the absorbance at 412 nm.

Liquid chromatography/Mass Spectrometric Analysis was performed on a TSQQuantum Triple quadrupole mass spectrometer with extended mass range(Thermo Electron, San Jose Calif.). Samples were chromatographed on aPRLP-S, 1000 A, microbore column (50 mm×2.1 mm, Polymer Laboratories,Shropshire, UK) heated to 75° C. A linear gradient from 30-40% B(solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile)was used and the eluant was directly ionized using the electrospraysource. Data were collected by the Xcalibur data system anddeconvolution was performed using ProMass (Novatia, LLC, New Jersey).Prior to LC/MS analysis, antibodies or antibody-drug conjugates (50 μg)are treated with PNGase F (2 units/ml; PROzyme, San Leandro, Calif.) for2 hours at 37° C. to remove N-linked carbohydrates.

Hydrophobic Interaction Chromatography (HIC) samples were injected ontoa Butyl HIC NPR column (2.5 μm, 4.6 mm×3.5 cm) (Tosoh Bioscience) andeluted with a linear gradient from 0 to 70% B at 0.8 ml/min (A: 1.5 Mammonium sulfate in 50 mM potassium phosphate, pH 7, B: 50 mM potassiumphosphate pH 7, 20% isopropanol). An Agilent 1100 series HPLC systemequipped with a multi wavelength detector and Chemstation software wasused to resolve and quantitate antibody species with different ratios ofdrugs per antibody.

Example 7 Conjugation of Antibodies and Anthracycline DerivativeDrug-Linker Intermediates

Generally, antibodies and anthracycline derivative drug-linkerintermediates are conjugated according to the methods of U.S. Pat. No.7,521,541; U.S. Pat. No. 7,498,298; US 2005/0276812; US 2008/0311134;and “NEMORUBICIN METABOLITE AND ANALOG ANTIBODY-DRUG CONJUGATES ANDMETHODS”, PCT/US2009/031199, filed 16 Jan. 2009, each of which areincorporated by reference.

Conjugates quantization analysis by in-gel fluorescence detection wasperformed using ProXpress CCD-based scanner (PerkinElmer). Instrumentexcitation and emission filters were set at 480/30 nm and 590/35 nmrespectively. Quantization analysis was performed using Profindersoftware provided with the instrument using the different amount ofstarting materials loaded on the gel as reference. Total loaded proteinwas then evaluated by Coomassie Blue protein staining

Example 8 Conjugation of Cysteine Engineered Antibodies and MaleimideDrug-Linker Intermediates

After the reduction and reoxidation procedures of Example 6, thecysteine engineered antibody is dissolved in PBS (phosphate bufferedsaline) buffer and chilled on ice. About 1.5 molar equivalents relativeto engineered cysteines per antibody of an anthracycline derivative witha thiol-reactive functional group such as: pyridine-disulfide, e.g.drug-linker intermediates 53 or; bromo-acetamido and maleimide, e.g.drug-linker intermediates 51 and 52, is dissolved in DMSO, diluted inacetonitrile and water, and added to the chilled reduced, reoxidizedantibody in PBS. After about one hour, an excess of maleimide is addedto quench the reaction and cap any unreacted antibody thiol groups. Thereaction mixture is concentrated by centrifugal ultrafiltration and thecysteine engineered antibody-drug conjugate is purified and desalted byelution through G25 resin in PBS, filtered through 0.2 μm filters understerile conditions, and frozen for storage.

By the procedures above, cysteine engineered antibody drug conjugateswere prepared: 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, and 113(Table 2). Each of the cysteine engineered antibodies comprisingantibody-drug conjugates 102, 103, 104, 105, 106, 107, 108, 109, 111,112, and 113 were the heavy chain (HC) A114C (Kabat) cysteine engineeredmutant (U.S. Pat. No. 7,521,541). For the trastuzumab antibody, theA114C mutant by the Kabat numbering scheme is the same as the A118Cmutant by the EU numbering scheme and the A121C mutant by the Sequentialnumbering scheme.

Cysteine engineered antibody drug conjugates with the maleimide caproyl(MC), valine-citrulline (vc), p-aminobenzyloxycarbamoyl (PAB), andauristatin drug (MMAE) drug-linker moieties (106, 111, and 112) wereprepared by the method of Example 3 of US 2008/0311134, and Examples 27and 29 of U.S. Pat. No. 7,498,298, which are incorporated by reference,with drug-linker intermediate MC-vc-PAB-MMAE:

Example 9 In Vitro Cell Proliferation Assay

Tumor cell lines breast carcinoma BT-474, SKBR-3, and MCF7 were obtainedfrom American Type Culture Collection.

The in vitro potency of antibody-drug conjugates was measured by a cellproliferation assay (FIGS. 8-29). The CellTiter-Glo® Luminescent CellViability Assay is a commercially available (Promega Corp., Madison,Wis.), homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670). This assay determines the number of viablecells in culture based on quantitation of the ATP present, an indicatorof metabolically active cells (Crouch et al (1993) J. Immunol. Meth.160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The substrate, Beetle Luciferin, is oxidatively decarboxylatedby recombinant firefly luciferase with concomitant conversion of ATP toAMP and generation of photons. Viable cells are reflected in relativeluminescence units (RLU). Data can be recorded by luminometer or CCDcamera imaging device. The luminescence output is presented as RLU,measured over time. Alternatively, photons from luminescence can becounted in a scintillation counter in the presence of a scintillant. Thelight units can be represented then as CPS—counts per second.

Efficacy of ADC were measured by a cell viability assay employing thefollowing protocol, adapted from CellTiter Glo Luminescent CellViability Assay, Promega Corp. Technical Bulletin TB288 and Mendoza etal (2002) Cancer Res. 62:5485-5488:

1. An aliquot of 50 μl of cell culture containing about 1000 or morecells, including:

HER2-expressing and CD22-expressing cells, in medium was deposited ineach well of a 96-well, opaque-walled, clear bottom plate.

2. ADC (50 μl) was added to triplicate experimental wells to finalconcentration of 10 μg/mL, with “no ADC” control wells receiving mediumalone, and incubated for 3 or more days.

3. The plates were equilibrated to room temperature for approximately 30minutes.

4. CellTiter-Glo Reagent (100 μl) was added.

5. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.

6. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.

7. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

Example 10 Pharmacokinetics—Serum Clearance and Stability

The disposition of antibody-drug conjugates in vivo is analyzed bymeasuring the serum concentrations of antibody and of drug conjugateafter a single intravenous bolus dose into Sprague-Dawley rats.Concentrations of antibody-drug conjugates bearing at least onecytotoxic drug are measured with an ELISA that used a extracellulardomain (ECD) protein for the capture and anti-anthracycline andhorseradish peroxidase (HRP) conjugated anti-mouse Fc antibody fordetection. Total antibody concentrations in serum were measured with anELISA that uses ECD for capture and anti-human-Fc HRP for detection,measuring antibody, both with and without conjugated anthracyclinederivative. The serum concentration-time data from each animal isanalyzed using a two-compartment model with IV bolus input, first-orderelimination, and macro-rate constants (Model 8, WinNonlin Pro v.5.0.1,Pharsight Corporation, Mountain View, Calif.).

Example 11 Animal Toxicity

A 12 day acute toxicity study in adolescent female rats (100-125 gms) isconducted by a single injection of antibody-drug conjugate at about 1-10mg/kg, and a control Vehicle at day 1. Injection of test article istypically administered as an intravenous bolus. Body weight is measureddaily. Clinical chemistry, serum enzymes and hematology analysis isconducted periodically. Toxicity signals included the clinicalobservation of weight loss.

Example 12 Tumor Growth Inhibition, In Vivo Efficacy Mouse Model

All animal studies were performed in compliance with NIH guidelines forthe care and use of laboratory animals and were approved by theInstitutional Animal Care and Use Committee at Genentech.

Efficacy studies were performed using SCID mice (Charles RiverLaboratories). The efficacy models for the studies exemplified in FIGS.30-32 were employed as described (Polson et al (2009) Cancer Res.69(6):2358-2364; Phillips et al (2008) Cancer Res. 68(22):9280-9290; US2008/0050310; US 2005/0276812), evaluating tumor volume after a singleintravenous dose. Transplant models were developed using tumors excisedfrom a mouse bearing an intraperitoneal tumor, then serially passagedinto recipient mice. For example, cells for implantation were washed andsuspended in HBSS (Hyclone) and inoculated subcutaneously into theflanks of female CB17 ICR severe combined immunodeficiency mice (7-16weeks of age from Charles Rivers Laboratories), in a volume of 0.2mL/mouse. To test the efficacy of the antibody drug conjugates in vivo,approximately several million cells per SCID mouse were inoculated onceand allowed to grow for about 10 to 60 days post-injection. When tumorvolumes reached 150-200 mm³ (typically Day 14 to Day 21 afterinoculation), the mice were segregated into sample groups of 9-10 miceper group and the tumor volume was determined in each mouse. When meantumor size reached the desired volume, the mice were divided into groupsof 8 to 10 mice with the same mean tumor size and dosed intravenouslyvia the tail vein with samples: ADCs, antibodies, or Vehicle. ADC doseswere either measured as the mass of the conjugated cytotoxic smallmolecule drug per surface area of the mouse, or as a constant mass ofADC per mass of the mouse (e.g., 5 mg of ADC/kg mouse). In general, thedrug loads on the antibodies in any given experiment were similar ornormalized, so these two measures could be considered equivalent. Tumorvolume was measured using calipers according to the formula: V(mm³)=0.5A×B², where A and B are the long and short diameters,respectively. Mice were euthanized before tumor volume reached 3000 mm³or when tumors showed signs of impending ulceration. Data collected fromeach experimental group were expressed as mean±SE.

1. An anthracycline derivative of formula (IIc)Ant-L-(Z)_(m)—X  (IIc) wherein Ant is selected from the structures:

where the wavy line indicates the attachment to L; L is a linkerselected from —CH₂O—, —N(R)_(m)(C₁-C₁₂ alkylene)-X¹, —N(R)—,—N(R)_(m)(C₁-C₁₂ alkylene)-, —N(R)_(m)(C₂-C₈ alkenylene)-,—N(R)_(m)(C₂-C₈ alkynylene)-, —N(R)_(m)(CH₂CH₂O)_(n)—, and thestructures:

where the wavy lines indicate the attachments to Ant and Z; and Z is anoptional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂ alkylene)-,and the structures:

X is a reactive functional group selected from maleimide, thiol, amino,bromide, p-toluenesulfonate, iodide, hydroxyl, carboxyl, pyridyldisulfide, and N-hydroxysuccinimide; R is H, C₁-C₁₂ alkyl, or C₆-C₂₀aryl; R¹ and R² are independently selected from an amino acid sidechain; Z¹ is selected from —S—, —CH₂C(O)—, —(CH₂CH₂O)_(n)CH₂C(O)—,—(CH₂CH₂O)—CH₂—and —(C₁-C₁₂ alkylene)-; m is 0 or 1; and n is 1 to
 6. 2.The anthracycline derivative of claim 1 selected from the structures:

wherein Z—X is selected from:


3. The anthracycline derivative of claim 2 having the structure:


4. The anthracycline derivative of claim 2 having the structure:


5. The anthracycline derivative of claim 1 having the structure:


6. The anthracycline derivative of claim 1 having the structure:


7. The anthracycline derivative of claim 1 having the structure:

where Z is C₁-C₁₂ alkylene.
 8. The anthracycline derivative of claim 7having the structure:


9. The anthracycline derivative of claim 1 having the structure:


10. The anthracycline derivative of claim 9 having the structure:


11. The anthracycline derivative of claim 9 having the structure:


12. The anthracycline derivative of claim 9 selected from thestructures:


13. The anthracycline derivative of claim 12 wherein Z¹ is —(C₁-C₁₂alkylene)-.
 14. The anthracycline derivative of claim 12 having thestructure:


15. The anthracycline derivative of claim 1 wherein R¹ and R² areindependently selected from hydrogen, methyl, isopropyl, isobutyl,sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃,—CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂,—(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂,—(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂,—(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl, and the following structures:


16. The anthracycline derivative of claim 1 selected from thestructures:


17. The anthracycline derivative of claim 1 selected from thestructures:


18. The anthracycline derivative of claim 1 selected from thestructures: